UC-NRLF 


B    3    TD5    IDA 


LIBRARY 

OF   THE 

UNIVERSITY  OF  CALIFORNIA. 

Class 


BIOLOGICAL 
LABORATORY   METHODS 


PLATE  I. 


BIOLOGICAL 


LABORATORY    METHODS 


BY 


P.    H.    MELL,  PH.D. 


DIRECTOR  OF  ALABAMA  EXPERIMENT  STATION, 

PROFESSOR  OF  GEOLOGY  AND   BOTANY,  ALABAMA    POLYTECHNIC 
INSTITUTE 


OF  THE    * 

I  UNIVERSITY  ) 

OF 


THE    MACMILLAN    COMPANY 

LONDON  :  MACMILLAN  &  CO.,  LTD. 
1902 

All  rights  reserved 


fiENERAl 


COPYRIGHT,  1902, 
BY  THE  MACMILLAN  COMPANY. 


Set  up  and  electrotyped  October,  1902. 


J.  S.  Gushing  &  Co.  -  Berwick  &  Smith 
Norwood  Mass.  U.S.A. 


CONTENTS 


PAGE 

INTRODUCTION xi 

CHAPTER  I 
THE  MICROSCOPE  .        .        .        .       ,        ...        .        .        .       .        i 

..     '         .",?.   i* 
CHAPTER   II 

OCULARS  AND  OBJECTIVES 21 

CHAPTER  III 
APPARATUS  AND  ACCESSORIES .45 

CHAPTER  IV 
PREPARATION  OF  THE  TISSUE  FOR  MOUNTING 66 

CHAPTER  V 
IMBEDDING  METHODS 83 

CHAPTER  VI 
STAINS,  THEIR  PREPARATION  AND  USE 105 

CHAPTER  VII 
MOUNTING  THE  TISSUE  FOR  PRESERVATION 127 

CHAPTER  VIII 
How  TO  MAKE  DRAWINGS  OF  THE  SECTIONS  OF  TISSUE     .        .        .132 

CHAPTER   IX 

PHOTO-MICROGRAPHS.    THE  APPARATUS 141 

v 


127799 


vi  CONTENTS 

CHAPTER  X 

PAGE 

MAKING  THE  PHOTO-MICROGRAPHS  (CONTINUED)         .        .        .        .156 

CHAPTER   XI 

PHOTO-MICROGRAPHS  (CONTINUED)  —  SUNLIGHT  AND  ELECTREC  LIGHT, 
OXY-HYDROGEN,  ACETYLENE,  ClTY  GAS,  AND  OlL  LAMPS,  AND 
HOW  TO  USE  THEM 165 

CHAPTER   XII 
PHOTO-MICROGRAPHS  (CONTINUED)  '• —  PHOTOGRAPHIC  DRY  PLATES  AND 

MAKING  THE  NEGATIVES         .         .         .         .         .        .  ,  .     173 

CHAPTER   XIII 
MAKING  THE  POSITIVES 186 

CHAPTER   XIV 

APPARATUS  AND  METHODS  IN  THE  STUDY  OF  BACTERIOLOGY      .        .    200 

CHAPTER   XV 
BLEACHING,  DECALCIFICATION,  INJECTION,  MACERATION       .        .        .    225 

CHAPTER  XVI 

POLARIZATION  OF  LIGHT  AND  ITS  APPLICATION  TO  BIOLOGICAL  IN- 
VESTIGATIONS   252 

CHAPTER  XVII 
USEFUL  FORMULA  AND  TABLES    . 260 

APPENDIX 
THE  ARRANGEMENT  OF  THE  LABORATORY  AND  ITS  FURNITURE  .        .    305 

INDEX 315 


ILLUSTRATIONS 

PAGE 

Apparatus  for  removing  Air  Bubbles  from  Slides 295 

Anaerobic  Apparatus,  Novy's 222 

Anaerobic  Culture,  Kasparec's 221 

Anaerobic  Culture,  Wright's 281 

Autoclave .  207 

Bleaching,  Marsh's  Chlorin  Method 227 

Box  for  Lantern  Slides 195 

Camera,  Kodak  Cartridge 142 

Camera,  Large  Photo-micrographic,  Zeiss's 163 

Camera,  Small  Photo-micrographic,  Zeiss's      .         .         .         .  160-161 

Camera  Lucida,  Bausch  &  Lomb     .         .         .         .         .         .         .         .135 

Camera  Lucida,  Section,  Abbe's 134 

Camera  Lucida,  Abbe's 133 

Chromatic  Aberration 5 

Coddington  Lens    .         .         .         .         .         .         .  .       .         .         .         .  2 

Counting  Apparatus 223 

Cover-glass  Circles 128 

Cover-glass  Squares 128 

Cover-glass  Gauge,  B.  &  L 29 

Cover-glass  Gauge,  Zeiss's        .........  30 

Dehydrating  Apparatus,  Thomas's 59 

Developing  Tray 151 

Diaphragm  Shutter,  Iris 148 

Dissecting  Microscope,  Zeiss's 3 

Dissecting  Needles 55 

Dissecting  Scalpel 56 

Dissolving  Key  for  Lantern 198 

Drawing-table,  Bernhard's 136 

Drawing-table,  B.  &  L.     Modification  of  Bernhard's        .         .         .         .  137 

Electric  Lamp  for  Lantern,  Colt's 168 

Eyepiece  Micrometer 27 

Film  Cartridge 145 

Fixing -bath,  Gennert's  Universal 151 

vii 


Vlli  ILLUSTRATIONS 

PAGE 

Flasks,  Erlenmeyer's 59 

Flasks,  Koch's 59 

Forceps 54 

Freezing  Attachment 98 

Glass  Benches 57 

Graduate i^i 

Hanging-drop  Culture p       .         .         .219 

Illuminating  Apparatus,  Abbe's 16 

Incubator,  Laboratory 208 

Injection  Apparatus,  Sterling's  Constant  Pressure 235 

Laboratory  Tables 208-209 

Lamp,  Carbutt's  Multum  in  Parvo 149 

Lamp,  Acetylene 172 

Lantern,  Colt's  Criterion  with  Electric  Lamp  and  Microscope  Attachment  197 

Lens  of  Crown  Glass  and  Flint  Glass 6 

Mat  for  Lantern  Slide      ..........  194 

Microscope,  Section  of 9 

Microscope,  Continental  BB 7 

Microscope,  Stand  la  with  Mechanical  Stage,  Zeiss's       .       Frontispiece 

Microscope,  Leitz's II 

Microscope,  Spencer's 13 

Microtome,  Hand 46 

Microtome,  Automatic 47 

Microtome,  Minot's  Rotary     .         .         .         ...         .         .         .48 

Microtome,  Minot's  Precision 50 

Microtome,  Ribbon  Carrier 49 

Microtome,  Reichert's .51 

Microtome  Knives 52 

Microtome  Knife-holder          .         .         . 52 

Microtome,  Freezing,  Section  of 95 

Microtome,  Jung's  Freezing 97 

Needle-holders  and  Needles 55 

Negative  Washing-bath  .         . 152 

Negative  Drying-rack 153 

Nivellating  Apparatus 220 

Nose-piece 19 

Objectives 31 

Objective,  Section  showing  Oil  Immersion 32 

Objective,  Section  of  Apochromatic 37 

Oculars,  Compensating 22 

Oculars,  Continental 21 


ILLUSTRATIONS  ix 

PAGE 

Oculars,  Huyghenian 21 

Oculars,  Micrometer 27 

Oculars,  Filar  Micrometer 27 

Oculars,  Projection 26 

Paraffin  Imbedding  Box 64 

Petri  Dish 58 

Pipette 58 

Polarized  Light       \ 253 

Polarizers,  Zeiss's     . 254 

Pressure  Gauge 171 

Printing-frame 153 

Ray  Filter .         .         .  150 

Retouching-frame 153 

Roll-holders,  Cartridge    .         . 144 

Rheostat,  Colt's .         .         .         .  169 

Scissors,  Dissecting 54 

Section  Lifters 57 

Sliding  Objective  Changers,  Zeiss's 19 

Slides,  American 128 

Slides,  German 128 

Spherical  Aberration 4 

Staining-jar,  Naples 109 

Staining-dish,  Moore's 109 

Sterilizer,  Hot-air,  Lautenschlaeger's 204 

Sterilizer,  Hot-air,  B.  &  L 203 

Sterilizer,  Steam,  Arnold's .  205 

Sterilizer,  Steam,  Section  of  Arnold's 206 

Swing-out  Condenser,  Zeiss's  .         .         .         .         .         .         .         .         .17 

Swing-out  Condenser  attached  to  Microscope,  Zeiss's       .         .         .         .18 

Syringe,  Stroschein's  Injection 235 

Syringe  with  Cannulas 234 

Thermo-regulator    .         .         .         .         .     ' 210 

Tripod  Case 143 

Tripod,  Folding 143 

Turn-table .60 

Water-bath,  Lillie's 63 

Water-bath,  Reeves's .62 

Water-bath,  Miller's 61 

Watch-glasses 58 

Wire  Baskets  for  Culture  Tubes 209 


INTRODUCTION 

THE  establishment  of  the  Experiment  Stations  in  the  several 
states  of  the  Union  in  connection  with  the  colleges  has  given 
such  a  strong  impetus  to  scientific  research  that  no  institution 
is  considered  to  be  well  equipped  unless  it  has  a  laboratory  for 
biological  investigation,  and  a  course  of  work  in  this  laboratory 
made  a  part  of  the  curriculum  of  the  college.  The  demand  is 
therefore  becoming  greater  each  year  for  suitable  text-books 
which  will  give  full  and  clear  instructions  concerning  the  use 
of  the  microscope  and  the  other  instruments  and  methods  re- 
quired in  these  laboratories.  It  is  true  there  are  excellent 
works  in  existence  which  treat  of  the  microscope  and  the 
methods  in  vogue  among  investigators,  but  these  books  are 
generally  in  such  shape  as  to  render  them  suitable  simply  for 
works  of  reference.  For  they  are  either  too  voluminous  or  de- 
voted to  only  one  or  a  few  of  the  many  subjects  it  is  required 
of  a  student  in  biology  to  know,  and  they  are  thus  rendered 
unsuitable  for  text-books  ;  and  they  take  up  the  subject  in  many 
instances  where  the  beginner  has  insufficient  knowledge  of  the 
topic  to  follow  the  author.  A  text-book  for  students,  particu- 
larly in  practical  work,  should  begin  at  the  beginning  and  treat 
of  all  matter  relating  to  the  subject  in  simple  language,  and  in 
sufficient  detail  to  convey  to  the  mind  of  the  student  a  clear 
comprehension  of  the  work  to  be  required  at  his  hands. 

The  editor  of  the  Journal  of  Applied  Microscopy  in  a  recent 
issue  of  this  most  excellent  paper  has  clearly  stated  the  matter 
when  he  says  :  "  Books  which  begin  at  the  beginning,  and  which 
deal  with  facts  in  a  perfectly  scientific,  accurate  manner,  and 
yet  with  the  utmost  simplicity,  which  will  aid  the  beginner 
without  confusing,  which  will  develop  his  powers  of  observation 


Xli  INTRODUCTION 

without  detracting  from  the  thing  itself,  and  which  will  tell  him 
the  what,  where,  and  how  of  microscopical  work,  will  be  appre- 
ciated by  the  thinking  laity." 

In  the  preparation  of  this  book  the  author  has  also  been 
guided  by  what  seemed  to  be  the  average  advancement  of  the 
students  who  report  from  year  to  year  for  admission  to  the 
scientific  courses  in  the  Alabama  Polytechnic  Institute.  The 
topics  treated  in  the  following  pages  have  not  been  greatly 
elaborated,  but  the  discussions  have  been  conducted  far  enough 
to  give  something  more  than  simply  suggestions  and  outlines. 
It  is  expected  that  the  student  will  build  only  the  foundation 
in  the  study  of  this  book,  but  it  is  hoped  by  the  author  that  the 
information  contained  herein  will  be  the  incentive  for  inducing 
the  student  to  extend  his  investigations  in  the  domain  of  bio- 
logical science  until  he  becomes  proficient  in  research  and  is 
able  to  contribute  to  the  knowledge  of  the  subject. 

In  writing  this  book  liberal  use  has  been  made  of  the  stand- 
ard works  relating  to  microscopy  and  to  histology,  and  much 
valuable  information  has  been  secured  from  the  papers  and 
periodicals  devoted  to  the  science  of  biology. 

The  following  firms  have  placed  the  author  under  obliga- 
tions for  permission  to  use  electroplates  from  which  some  of 
the  illustrations  have  been  produced :  Bausch  &  Lomb  Opti- 
cal Company;  Carl  Zeiss  Company;  Eastman  Kodak  Com- 
pany ;  Buffalo  Dental  Manufacturing  Company ;  and  Rochester 
Optical  Company. 

P.  H.  MELL. 

AUBURN,  ALABAMA, 
October,  1902. 


BIOLOGICAL 
LABORATORY   METHODS 


BIOLOGICAL  LABORATORY  METHODS 


CHAPTER   I 

THE    MICROSCOPE 

THE  microscope,  an  essential  instrument  in  every  laboratory, 
should  be  well  made,  out  of  the  best  material,  to  enable  the  student 
to  perform  satisfactory  work  and  secure  accurate  results. 

There  is  no  economy  in  purchasing  a  "cheap"  microscope, 
but  great  care  and  experienced  judgment  should  be  exercised  in 
providing  each  student  with  a  first-class  instrument  made  by  the 
most  reliable  manufacturer.  This  important  fact  being  well  under- 
stood and  appreciated,  it  then  becomes  a  matter  largely  controlled 
by  taste  and  the  character  of  the  work  which  instrument,  from  the 
list  of  less  than  a  dozen  responsible  opticians,  it  is  advisable  to 
purchase.  In  this  country  and  abroad  excellent  microscopes  are 
made  by  such  houses  as  Bausch  &  Lomb  Optical  Company,  Zent- 
mayer,  Zeiss,  Leitz,  and  others. 

The  cost  of  the  microscope  will  increase  in  proportion  to  the 
delicacy  of  the  work  required,  the  minuteness  of  the  object  under 
investigation,  and  the  advancement  of  the  student  in  histology. 
So  that,  after  determining  upon  the  pattern  of  stand,  the  most 
important  question  to  be  answered  is  what  kind  and  number  of 
objectives  and  oculars  are  required.  As  a  general  rule  it  is  best  to 
begin  with  two  or  three  objectives  with  oculars  to  match,  and  add 
to  the  list  as  the  needs  demand. 

Each  student  should  be  provided  with 

1.  A  Simple  or  Dissecting  Microscope. 

2.  A   Compound  Microscope. 


2  BIOLOGICAL  LABORATORY   METHODS 

The  dissecting  microscope  is  a  convex  glass  lens  mounted  in  a 
metal  or  rubber  holder  and  attached  to  a  convenient  stand  with 
rack  and  pinion  adjustment,  mirror  and  stage,  so  that  the  object 
may  be  placed  under  the  lens,  a  steady  focus  secured,  and  the 
hands  left  free  to  manipulate  the  dissecting  instruments  with  the 
least  degree  of  inconvenience.  The  image  produced  by  this  in- 
strument is  not  inverted  as  it  is  in  the  compound  microscope, 
and  hence  the  dissection  can  be  performed  with  much  greater 
ease. 

For  a  long  time  the  single  glass  employed  in  the  earlier  history 
of  this  instrument  produced  spherical  aberration  and  aberration  of 
refrangibility  to  such  a  great  degree  it  was  found  necessary  to  re- 
place the  single  lens  with  what  Wollaston,  the  inventor,  termed 
doublets.  The  two  lenses,  or  doublets,  in  Wollaston's  instrument 
are  so  adjusted  in  relation  to  each  other  that  they  act  together 
as  a  single  glass.  They  are  plano-convex  and  are  separated  by 
means  of  a  diaphragm.  Besides  overcoming,  to  a  large  extent, 
spherical  aberration,  by  the  use  of  two  glasses  with  a  diaphragm 
interposed,  the  doublets  also  increase  the  distance  between  the 
lens  and  the  object,  thus  permitting  more  freedom  in  the  manipu- 
lation of  the  dissecting  instruments. 

Achromatic  triplets  may  also  be  used  on  the  microscope,  thus 
giving  a  large,  flat  field  and  a  beautiful  definition. 


FIG.  i.  —  Coddington  Magnifier. 

Coddington  magnifiers  (Fig.  i)  are  convenient  for  field  work 
and  are  highly  prized  by  all  practical  botanists.  The  figure  rep- 
resents an  aplanatic  triplet  lens  which  is  achromatic.  It  is  com- 
posed of  two  flint  lenses  between  which  is  cemented  a  thick  crown 


THE   MICROSCOPE  3 

glass.  The  magnification  is  about  ten  diameters,  and  the  image  is 
free  from  distortion. 

A  most  excellent  dissecting  microscope  is  made  by  the  Zeiss 
Optical  Company  of  Germany  and  is  illustrated  in  Fig.  2.  The 
manufacturers  in  describing  this  instrument  say  :  — 

"The  stage  consists  of  a  large  metal  frame  (10  x  10  cm.  = 
4  X  4  in.),  to  which  is  attached  wooden  folding  hand-rests;  ad- 
justment by  rack  and  pinion,  plane  and  concave  mirrors  with 


FIG.  2.  — Zeiss  Dissecting  Microscope. 

universal  motions,  to  which  a  piece  of  white  paper  may  be  at- 
tached by  means  of  a  ring  so  as  to  produce  with  low  magnifica- 
tions diffuse  illumination. 

"  The  triple  dissecting  system  "  which  consists  "  of  three  achro- 
matic lenses  (objectives)  and  an  achromatic  concave  eyepiece, 
forms  a  suitable  combination  for  dissecting  and  '  teasing '  small 
objects  on  a  slip  or  in  a  watch-glass ;  it  may  be  fixed  in  the 
ordinary  lens-holder  P,  and  a  black  metal  plate  with  stage  open- 
ing of  14  mm.  diameter,  which  can  be  closed  below  by  a  black  or 
white  disk,  may  be  placed  under  the  object  into  a  recess  provided 
in  the  stage-frame. 

"  For  examining  large  objects,  particularly  living  aquatic  ani- 
mals, the  aplanatic  lenses  will  be  found  useful.  These  glasses 


4          BIOLOGICAL  LABORATORY  METHODS 

are  composed  of  three  cemented  lenses,  giving  relatively  long 
focal  distances  with  large  flat  field.  They  fit  into  a  separate 
arm  LR,  which  may  be  inserted  into  the  ordinary  lens- support 
at  L,  and,  by  this  arrangement,  the  whole  of  the  stage  can  be 
scanned. 

"  In  this  case  the  metal  stage  is  replaced  by  a  glass  plate,  and 
the  interchangeable  white  and  black  disks  serve  as  convenient 
means  of  forming  a  white  and  black  ground. 

"  A  brass  plate  made  to  fit  the  frame  serves  as  a  support  for 
small  dissecting  dishes,  which  may  be  cemented  to  it  with 
paraffin." 

Spherical  and  chromatic  aberrations. — A  few  words  in  refer- 
ence to  this  subject  may  not  be  out  of  place  in  a  book  of  this 


V 

FIG.  3. —  Spherical  Aberration'. 

character  in  order  that  the  student  may  form  some  general  idea 
concerning  the  principles  upon  which  the  Wollaston  lens  is  con- 
structed. But,  for  a  full  and  detailed  account  of  refraction  of 
light  and  correction  of  spherical  and  chromatic  aberrations,  he  is 
referred  to  works  on  optics. 

Spherical  aberration,  so  common  in  single  lenses,  is  due  to  that 
property  a  glass  medium  has  of  refracting  the  rays  of  light  to  a 
greater  degree  near  the  circumference  than  toward  the  centre  of 


THE   MICROSCOPE 


5 


the  glass.  For  this  reason  many  foci  are  formed,  and  the  image 
becomes  confused  and  indistinct.  The  rays  near  the  axis  of  the 
lens  and  parallel  to  it  are  refracted  when  they  penetrate  the  lens 
and  are  brought  to  a  point  in  the  principal  focus ;  while  those 
rays  which  enter  the  lens  near  its  edges  are  refracted  more  and 
are  focussed  at  a  point  nearer  the  lens.  The  formation  of  these 
foci  by  the  same  pencil  of  light  is  termed  spherical  aberration. 
Optical  glasses  producing  this  defect  are  not  suitable  for  micro- 


FlG.  4.  —  Chromatic  Aberration. 

scopic  work,  and  the  trouble  must  be  overcome  before  objectives 
and  oculars  are  sent  out  by  the  manufacturers.  There  are  two 
ways  of  correcting  such  aberration  :  (i)  by  using  a  diaphragm 
which  partly  corrects  the  aberration  by  interrupting  the  scattered 
rays  coming  through  the  edges  of  the  lens  and  permitting  only 
those  rays  penetrating  the  lens  near  its  axis  to  produce  the 
image ;  (2)  by  so  grinding  the  lens  that  its  curvature  near  the 
edges  is  less  than  that  near  the  centre.  When  a  lens  is  corrected 
for  spherical  aberration  it  is  called  aplanatic. 

Chromatic  aberration  refers  to  the  decomposition  of  the  rays 
of  light  into  colors  when  they  pass  through  a  lens.  This  is  also 
a  serious  defect  which  must  be  corrected  as  far  as  possible  if  the 


6          BIOLOGICAL  LABORATORY  METHODS 

objective  is  expected  to  do  first-class  work.  It  is  a  well-known 
fact  to  physical  students  that,  of  all  the  colors  produced  by  a  ray 
of  light  which  is  refracted  through  a  prism,  the  violet  color  (its 
index  of  refraction  being  1.5466)  suffers  the  greatest  refraction, 
while  the  red  (its  index  being  1.5258)  at  the  other  limit  of  the 
spectrum  submits  to  the  least  refraction.  These  two  rays,  there- 
fore, after  separating  through  the  medium  of  the  glass,  meet  the 
principal  axis  of  the  lens  at  different  points,  the  violet  nearest 
the  glass,  at  v,  and  the  red  at  r,  farthest  removed  from  the  lens. 
A  screen  placed  at  ss  will  have  projected  on  it  a  red  ring  at 
those  points  where  the  red  ray  strikes  ;  and  if  the  screen  is 
removed  to  a  position  between  v  and  the  lens,  two  colored  rings 
will  be  formed,  the  outer  one  will  be  red  and  the  inner  one 
violet.  When  an  object  is  viewed  through  a  lens  giving  such 
results,  all  colors  of  the  rainbow  are  discernible,  and  the  image 
becomes  very  much  confused  and  indistinct,  as  it  is  with  lenses 
producing  spherical  aberration.  To  correct  chromatic  aberra- 
tion two  different  kinds  of  glasses  must  be  used  in  making  the 

lens.  One  is  crown  glass, 
or  silicate  of  potassium  and 
lime,  which  has  an  index  of 
refraction  of  about  1.52  and 
a  dispersive  power  of  0.036  ; 
the  other  glass  is  flint,  or 
silicate  of  potassium  and 


FIG.  5.-  Lens  of  Crown  and  Flint  Glass.        ^   wkh   an    in(kx  Q£    re_ 

fraction  of  about  1.63  and  a  dispersive  power  of  0.059.  These 
two  glasses  when  combined  have  the  power  of  so  acting  on  each 
other  that  when  a  ray  passes  through  them  it  is  not  only  re- 
fracted but  the  decomposition  of  light  produced  by  one  glass  is 
considerably  overcome  by  the  other,  and  the  colors  are  again 
recombined  into  white  light  and  brought  to  a  focus  with  more 
or  less  distinctness  in  the  image.  The  angles  of  these  lenses 
must  be  unequal,  in  order  that  the  dispersive  powers  may  be 
the  same.  The  usual  form  for  the  crown  glass  is  bi-convex 
and  that  for  flint  is  plano-convex.  They  are  cemented  together 


(Cut  one-half  actual  size.) 
GLASS  STAGE. 


(Cut  one-half  actual  size.) 

BBS— CONTINENTAL  MICROSCOPE. 
PLATE  II. 


8          BIOLOGICAL  LABORATORY  METHODS 

with  Canada  balsam.  A  lens  so  constructed  is  called  achro- 
matic. 

For  a  long  time  it  was  found  to  be  impossible  with  the  infor- 
mation possessed  by  the  opticians,  entirely  to  overcome  chro- 
matic aberration,  even  after  many  experiments  in  this  country 
and  abroad  had  produced  numerous  different  kinds  of  glasses  of 
the  flint  and  crown  varieties. 

Some  years  ago  Professor  Abbe  and  Dr.  Schott,  two  skilful 
experimenters  of  Jena,  Germany,  began  investigations  with  dif- 
ferent compounds  of  vitreous  materials  for  the  purpose  of  dis- 
covering some  method  for  correcting  chromatic  aberration  and 
other  defects  which  were  so  troublesome  to  the  optical  manufac- 
turers. They  succeeded  in  discovering,  by  means  of  elaborate 
and  expensive  experiments,  compounds  closely  resembling  glass, 
whose  properties  are  so  remarkable  that  a  lens  manufactured 
from  the  compound  for  the  microscope  gives  an  image  with 
marvellous  distinctness,  even  to  the  outer  limits  of  the  field. 
These  glasses  do  not  possess  silica  in  their  composition,  which 
we  found  to  be  the  case  with  the  flint  and  crown  glasses  in 
the  achromatic  lenses  above  described,  but  in  the  Jena  glass 
the  silicon  is  replaced  by  boron  in  the  flint  series  and  by 
phosphorus  in  the  crown  series.  Since  1889  the  Zeiss  Com- 
pany of  Jena,  Germany,  have  been  making  objectives,  eye- 
pieces, and  other  lenses  from  this  new  glass,  and  have  sup- 
plied other  optical  companies  in  different  parts  of  the  world 
with  the  compound,  so  that  now  the  best  microscopes  are  fur- 
nished with  objectives  made  of  the  new  composition.  The 
Zeiss  Company,  which  have  the  patent  rights  on  the  Abbe  and 
Schott  discoveries,  call  the  glasses  made  from  the  new  com- 
pound apochromatic. 

This  discovery  has  produced  material  of  certain  refractive 
and  dispersive  properties  which  make  the  glass  specially  valu- 
able for  objectives,  and  the  amount  of  light  concentrated  in  the 
image  is  greatly  increased  over  that  yielded  by  lenses  made  of 
flint  and  crown  glass.  The  apochromatic  objectives  made  of 
this  new  glass  are  much  more  expensive  than  the  ordinary  achro- 


THE  MICROSCOPE 


matic  objectives,  and  they 
require  in  their  manufac- 
ture greater  skill.  It  is 
quite  desirable  to  have  the 
laboratory  equipped  with 
these  apochromatic  objec- 
tives, and  some  delicate 
work  demands  their  use ; 
still  much  of  the  work  re- 
quired of  students  can  be 
well  performed  with  micro- 
scopes supplied  with  achro- 
matic objectives,  provided 
they  are  carefully  made  by 
the  best  manufacturers  of 
microscopes. 

The  compound  microscope. 
—  Figure  6  is  a  longitudi- 
nal section  of  the  instru- 
ment and  shows  that  it 
consists  of  two  tubes,  one 
sliding  in  the  other,  a  stage 
and  stand,  besides  the 
lenses,  called  oculars  and 
objectives.  The  inner  tube 
carries  in  the  upper  end  the 
ocular  OC,  and  to  the  lower 
end  of  the  outside  tube  is 
screwed  the  objective,  B. 
The  tubes  are  blackened 
on  the  inside  so  that  all 
reflection  of  the  light  rays, 
after  passing  through  the 
objective,  will  be 
obviated.  The  si — 
rays  of  light  com- 


FlG.  6.  —  Section  of  Compound  Microscope. 


10  BIOLOGICAL  LABORATORY   METHODS 

ing  from  the  object,  mounted  on  the  glass  slide  S,  pass  through 
the  objective  B  to  eyepiece  <9Cand  are  caught  by  the  eye  situated 
at  E.  The  real  image  is  formed  at  IM  and  the  virtual  image  at 
/'J/'.  This  virtual  image  is  projected  from  the  eye.  The  slid- 
ing of  the  tubes,  one  in  the  other,  permits  of  limited  elongation, 
so  that  the  length  may  be  adapted  to  the  character  of  the  objec- 
tive in  use.  For  instance,  the  objective  made  by  a  German 
optician  will  not  produce  satisfactory  results  when  used  on  an 
American  stand,  unless  especially  adjusted  for  it.  The  inner  or 
draw-tube  is  usually  graduated  into  inches  or  millimeters  to  en- 
able the  microscopist  to  secure  a  known  length  at  will.  At  the 
lower  end  of  the  draw-tube  is  a  screw  thread  to  hold  the  aux- 
iliary objective  when  the  apertometer  is  in  use,  determining  the 
numerical  and  angular  aperture  of  the  objectives.  The  objec- 
tives made  by  Zeiss  require  a  length  of  tube  of  160  mm.,  and 
this  length  is  "  reckoned  from  the  contact  surface  of  the  objec- 
tive thread  to  the  upper  end  of  the  body  on  which  the  eye- 
piece rests." 

The  following  table  is  given  to  show  how  much  this  matter  of 
tube-length  varies  with  different  manufacturers  throughout  the 
world :  — 

Reichert  of  Vienna 160  to  180  mm. 

Swift  &  Son  of  London 165  to  228*- 

Zeiss  of  Jena,  Germany          .         .         .         .         .  160  " 

Winkel  of  Gottingen    ......  220  " 

Ross  of  London 254  " 

Leitz  of  Wetzlar,  Germany 1 70  " 

R.  &  J.  Beck  of  London        .....  254  " 

Grunow  of  New  York 203  " 

Bausch  &  Lomb  of  Rochester,  N.Y.      .         .         .  160  to  216  " 

Spencer  &  Co.  of  Buffalo,  N.Y 1 60  to  235 

The  thickness  of  the  cover-glass,  on  slide  S  (Fig.  6),  has  a  great 
influence  also  over  the  results  secured  with  the  objective,  be- 
cause the  light  is  changed  in  its  character  and  direction  after 
passing  through  the  cover-glass,  and  many  rays  will  be  lost 
unless  the  proper  adjustment  is  made  in  the  objective.  It  is 


V 


w 


PLATE  III.—  LEITZ  MICROSCOPE. 


OF  THE 

UNIVERSITY 


12  BIOLOGICAL   LABORATORY   METHODS 

best,  therefore,  to  use  a  cover-glass  gauge,  when  one  of  the 
higher  series  of  objectives  is  on  the  microscope,  and  mount  with 
cover-glasses  of  an  estimated  thickness. 

Another  way  of  determining  the  thickness  of  the  cover-glass 
is  recommended  by  Zeiss  as  follows :  "  The  divisions  on  the 
milled  head  of  the  micrometer-screw  of  Stands  la-IV  (see  Fron- 
tispiece) furnish  a  means  for  exactly  registering  the  vertical 
movements  of  the  tube.  In  our  present  stands  each  division 
corresponds  to  an  elevation  or  depression  of  the  tube  in  the 
direction  of  the  optical  axis  of  o.oi  mm.,  the  half -divisions  repre- 
senting 0.005  mm-  By  this  means  measurements  of  thickness 
may  be  made  with  a  considerable  degree  of  accuracy.  The 
upper  and  lower  surfaces  of  the  object  are  successively  focussed, 
and  the  amount  read  off  on  the  milled  head  by  the  fixed  index. 
In  doing  this,  care  must  be  taken  to  make  both  adjustments  by 
a  rotation  of  the  screw  in  the  same  direction.  The  thickness  of 
an  object  in  air  is  then  equal  to  the  difference  between  the  two 
readings. 

"  Similarly,  the  thickness  of  any  other  substance  may  be 
measured  by  this  method.  The  estimation  of  the  thickness  of 
a  cover-glass,  for  instance,  is  best  done  as  follows :  with  a 
medium-power  dry  lens  and  eyepiece,  using  central  illumination, 
focus  the  upper  and  lower  surfaces  of  the  cover-glasses  of  known 
thickness  —  e.g.  the  covers  of  an  Abbe  test-plate  —  and  note  the 
apparent  thickness  so  obtained.  A  comparison  of  this  with  the 
known  true  value  gives,  once  for  all,  the  coefficient  for  reducing 
to  their  true  values  measurements  of  any  other  covers,  made 
with  the  same  objective  under  precisely  similar  conditions. 
Roughly  speaking,  this  coefficient  is  equal  to  1.52  (the  refractive 
index  of  glass).  The  thickness  of  a  section  is  estimated  in  a 
similar  manner." 

Besides  the  lengthening  of  the  tube,  above  indicated,  the  instru- 
ment is  also  provided  with  an  adjustment  which  permits  the 
raising  and  lowering  of  the  entire  tube-system  and  thus  bringing 
the  object  into  focus.  This  movement  is  under  the  control  of 
two  mechanisms :  one  called  the  coarse  adjustment  and  the  other 


(Cut  one-half  actual  size.; 
CONTINENTAL  FORM  NO. 

PLATE  IV. 


14  BIOLOGICAL  LABORATORY   METHODS 

the  fine  adjustment.  The  first  is  made  by  means  of  rack 
and  pinion  movement.  The  second  is  accomplished  by  turn- 
ing the  milled-head  micrometer-screw,  which  is  generally  grad- 
uated. 

In  the  cheaper  forms  of  instruments  the  rack  and  pinion  are 
wanting  and  the  coarse  adjustment  is  made  by  sliding  the  tube- 
system  up  and  down  by  a  twisting  motion  with  the  thumb  and 
finger. 

The  stand.  —  The  base  of  the  stand  should  be  made  of  strong 
material,  and  should  maintain  a  firm  and  steady  position  even 
when  the  tube  of  the  instrument  is  inclined  at  any  angle.  The 
Continental,  and  some  of  the  American  stands  also,  have  bases 
in  the  form  of  horseshoes,  while  many  of  the  other  makes  con- 
sist of  a  three-pronged  base. 

The  stage  occupies  a  position  on  the  microscope  just  under- 
neath the  tube  and  is  firmly  fixed  to  the  stand.  In  its  most  sim- 
ple form  it  consists  of  a  metal  table,  sometimes  rectangular  and 
at  other  times  round,  with  a  hole  through  the  centre  for  the 
passage  of  light  from  the  illuminating  system  beneath.  On  the 
stage  are  two  spring-clips  to  be  used  in  holding  the  slide  well 
clamped  to  the  stage.  Diaphragms  are  fitted  to  the  opening  in 
the  stage  to  control  the  quantity  of  light. 

The  mechanical  stage  placed  on  top  of  the  simple  stage  con- 
tains the  mechanism  necessary  for  producing  the  required  move- 
ments to  bring  all  portions  of  the  object  readily  into  view.  "  The 
available  lateral  movement  measures  50  mm.,  and  the  stage  moves 
through  35  mm.  in  the  direction  from  front  to  back.  The 
amounts  are  read  by  vernier  and  scales.  The  mechanical  stage 
(see  Frontispiece)  is  so  solidly  constructed  that  possessors  of  a 
stand  fitted  with  it  may  well  dispense  with  the  plain  vulcanite 
stage.  The  mechanism  (LK)  which  serves  for  the  lateral  move- 
ment of  the  object  lifts  off  after  unscrewing  the  fixing  screw  L, 
whereby  the  solid  lower  part  of  the  stage  becomes  free.  By 
means  of  the  milled  head  W  this  lower  part  of  the  stage  can  be 
made  to  move  in  a  forward  and  backward  or — if  the  whole  stage 
be  rotated — in  any  other  desired  direction." 


THE  MICROSCOPE  15 

How  to  use  the  mechanical  stage.  —  The  Bausch  &  Lomb  Opti- 
cal Company  in  this  country  and  the  Zeiss  Company  in  Germany 
manufacture  the  best  form  of  mechanical  stage.  The  Zeiss 
pattern  is  shown  in  the  Frontispiece.  This  stage  permits  of 
several  movements  in  making  measurements  of  objects.  The 
milled-head  screw  K  gives  a  movement  from  right  to  left,  and 
W  turns  the  stage  in  a  direction  at  right  angles  to  K.  There 
are  two  millimeter  scales  on  the  stage ;  one  is  shown  in  the  illus- 
tration just  under  the  milled-head  screw  K,  and  the  other  is  to  the 
extreme  left  of  the  stage  and  not  visible  in  the  illustration.  One 
is  at  right  angles  to  the  other,  thus  allowing  measurements  in 
two  directions.  A  vernier  slides  on  each  of  these  scales,  thus 
giving  a  means  for  delicate  measurements.  The  screw  L  is 
used  for  firmly  clamping  the  stage.  By  the  use  of  this  mechani- 
cal stage  lengths  and  breadths  can  be  measured  with  great 
degree  of  accuracy. 

The  illuminating  apparatus.  —  This  is  an  important  part  of  the 
microscope  and  should  be  well  and  accurately  made.  It  is  situ- 
ated beneath  the  stage.  The  simplest  form  is  a  circular  mirror 
with  two  reflecting  surfaces,  and  so  mounted  as  to  allow  of  the 
movement  necessary  to  catch  the  light  from  any  direction  and  re- 
flect it  through  the  stage  upon  the  object.  The  diameter  ought 
to  be  at  least  2  inches,  so  that  an  ample  amount  of  light  can 
be  reflected  by  it. 

There  are  two  reflecting  surfaces,  one  plane  and  the  other 
concave.  The  first  is  adapted  to  the  use  of  lower  power-objec- 
tives, because  the  light  is  reflected  with  initial  intensity.  The 
concave  mirror  concentrates  the  rays  and  gives  a  brighter  sur- 
face on  the  stage  than  that  produced  by  the  plane  surface,  and 
it  is  used  with  the  higher  powers,  except  when  the  condenser  is 
in  place.  The  points  to  be  observed  by  the  student  in  securing 
the  best  results  with  the  reflector  are :  — 

1.  Central  illumination. 

2 .  A  uniform  and  even  illumination  over  the  entire  field. 

3.  Not  too  intense  light. 


i6 


BIOLOGICAL  LABORATORY  METHODS 


The  control  of  the  quantity  of  light  may  be  had  by  the  judi- 
cious use  of  diaphragms,  the  rule  being  to  reflect  only  enough 
light  clearly  to  define  the  object  and  avoid  straining  the  eyes. 

How  to  use  the  condenser.  —  Professor  Abbe  has  probably  done 
more  to  develop  this  important  part  of  the  microscope  than  any 


FIG.  7. —  Abbe's  Illuminating  Apparatus. 

other  party.  The  essential  features  of  the  Abbe  condenser  are 
to  collect  the  rays  from  the  reflector  in  a  system  of  short  focus 
and  concentrate  them  in  an  illuminated  cone  of  large  aperture 
which  has  its  focal  point  in  the  plane  where  the  object  is  situated. 
This  condenser  consists  of  (i)  the  cylinder  for  carrying  the  iris 
diaphragm  and  (2)  the  swing-out  condenser. 


THE  MICROSCOPE  I/ 

The  use  of  this  instrument  must  be  governed  entirely  by  the 
character  of  objects  under  examination.  The  full  aperture  of 
the  illuminating  cone  is  only  adapted  to  objects  deeply  stained 
and  of  fine  granular  condition  in  connection  with  objectives 
of  large  aperture.  In  all  other  cases  the  condensers  must  be 
stopped  down. 

This  instrument  will  also  permit  of  dark  ground  illumination 
by  moving  the  diaphragm  eccentrically  with  the  rack  and  pinion 
attached  to  the  carrier,  thus  cutting  off  the  central  rays  and  pro- 
ducing oblique  illumination. 


FIG.  8.  —  Zeiss'  Swing-out  Condenser. 

The  condenser  is  so  mounted  that  the  system  may  be  made 
to  approach  or  recede  from  the  stage,  and  in  this  way  the  degree 
of  illumination  is  kept  under  perfect  control.  Between  the  con- 
denser and  the  reflector  is  an  iris  diaphragm,  which  is  very 
essential  to  the  system  in  reducing  or  enlarging  the  cone  of 
light.  This  diaphragm  is  controlled  by  means  of  a  pin  d  (Fig.  7). 
In  this  illustration,  a  is  the  condenser  system  of  1.20  numerical 
aperture  ;  b  is  the  condenser  system  of  1.40  numerical  aperture  ; 
c  is  the  cylinder  diaphragm  ;  and  e  is  the  milled  head  for  throw- 
ing the  diaphragm  out  of  centre.  The  "  swing-out  condenser  " 
is  shown  more  in  detail  in  Fig.  8.  It  is  thus  described 
by  the  manufacturer :  "  The  condenser  system  is  by  a  suitable 
c 


i8 


BIOLOGICAL  LABORATORY  METHODS 


mechanism  so  connected  to  the  sleeve  enveloping  it  that,  after 
swinging  the  diaphragm  carrier  D  aside  (toward  the  right  of 
the  observer),  it  may  by  means  of  the  lever  H  projecting  from 
under  the  stage  be  swung  downward  about  the  axis  Q  and  to 
the  left  about  the  pivot  Z.  When  the  condenser  is  not  being 
used,  the  width  of  the  illuminating  cone  is  regulated  by  means  of 


FIG.  9.  —  Swing-out  Condenser  attached  to  Microscope. 

the  iris  cylinder  diaphragm,  which  is  permanently  attached  to 
the  apparatus  and  is  actuated  by  the  small  pin  K  seen  on  the 
right.  The  iris  diaphragm  is  so  shaped  that  the  edge  of  its 
smallest  opening  closely  approaches  the  object  slide." 

The  iris  diaphragm  in  the  sub-stage  is  the  best  contrivance 
for  controlling  the  light  of  any  diaphragm  now  known.  There 
are  many  advantages  in  its  use,  among  which  may  be  mentioned 


THE  MICROSCOPE 


the  time  and  inconvenience  saved  in  substituting  one  metal  disk 
for  another,  as  is  required  in  the  old  system ;  the  gradual  exclu- 
sion of  the  rays  as  the  iris  is  contracted,  thus  producing  marked 
effects  in  the  ap- 
pearance of  the 
object. 

The  revolving 
nose-piece.  —  This 
is  a  mechanical 
device  for  enabling 
the  manipulator  to 
rapidly  and  con-  FIG.  ^-Revolving  Nose-piece. 

veniently  change  from  one  objective  to  another.     The  nose-piece 
(Fig.  10)  is  screwed  to  the  lower  end  of  the  tube  and  is  so  con- 


FIG.  ii.  FIG.  12. 

Zeiss*  Sliding  Objective  Changers. 


2O  BIOLOGICAL  LABORATORY  METHODS 

structed  as  to  hold  two,  three,  or  four  objectives  at  the  same 
time.  By  revolving  the  instrument  each  objective  is  imme- 
diately brought  into  use,  requiring  but  little  adjustment  of  the 
microscope  to  bring  out  the  image  clear  and  distinct. 

Sliding  objective  changers.  —  Dr.  Roderick  Zeiss,  of  Germany, 
has  devised  an  apparatus  (Figs,  n  and  12)  which  is  considered 
by  most  microscopists  to  be  superior  to  the  revolving  nose- 
piece.  This  instrument  is  called  the  sliding  objective  changer. 
The  appliance  consists  of  two  parts :  (i)  the  tube-slide ;  (2)  the 
objective  slide.  The  first  is  permanently  screwed  to  the  lower 
end  of  the  tube,  in  the  same  manner  explained  with  the  nose- 
piece.  The  lower  part  of  the  tube-slide  contains  a  grooved 
plane  inclined  to  the  optic  axis  of  the  microscope.  The  objec- 
tive slide  contains  a  similar  grooved  plane  inclined  also  to  the 
optic  axis,  and  when  the  objective  is  pushed  into  working  posi- 
tion the  inclined  planes  permit  the  lens  to  approach  gradually 
toward  the  slide  containing  the  object  without  danger  of  break- 
ing the  glasses.  This  sliding  objective  changer  is  adapted  and 
corrected  to  fit  certain  objectives  and  microscopes,  but  it  may 
be  altered  to  suit  any  standard  microscope  and  lens  by  simply 
adjusting  the  screws,  which  move  at  right  angles  to  each  other 
so  that  centring  may  be  accomplished  in  either  direction. 


CHAPTER   II 

OCULARS   AND    OBJECTIVES 

The  oculars.  —  There  are  two  kinds  of  oculars,  or  eyepieces, 
which  are  termed  positive  and  negative.  The  first  comprises 
those  glasses  in  which  the  real  inverted  image  is  made  outside 
the  ocular,  while  the  latter  have  the  image  produced  within  the 
glass.  The  positive  eyepiece  is  a  simple  magnifier  and  may  be 
used  in  place  of  the  ordinary  hand-glass ;  the  negative  lens 
cannot  be  so  used. 

The  most  important  oculars  are  :  — 


FIG.  13.  — Huyghenian  Ocular.    FIG.  14.  — Huyghenian  Ocular.     FIG.  15.  — Section  of 
American  Pattern.  Continental  Pattern.  Ocular. 

i.  Huyghenian  oculars  (Figs.  13  and  15).  —  These  consist  of 
two  plano-convex  lenses,  one  at  each  end  of  the  brass  tube. 
The  upper  lens  has  its  plane  surface  toward  the  observer,  and 
the  lower  lens  has  its  convex  surface  toward  the  object.  The 
upper  is  called  the  eye-glass,  and  the  lower  is  known  as  the  field 
or  collecting  glass.  Between  these  lenses  is  placed  a  diaphragm 

21 


22 


BIOLOGICAL  LABORATORY   METHODS 


00 


near  the  focus  of  the  eye-glass. 
This  ocular  belongs  to  the 
negative  type  and  is  used  in 
most  microscopes  made  on 
the  American  and  Continental 
plans.  There  are  two  kinds, 
viz. :  — 

a.  The   type  which  is  gen- 
erally used  on  American  micro- 
scopes (Fig.  13). 

b.  The     Continental    form, 
which  is  used  on  all  German 
and  French  instruments   (Fig. 

*5). 

2.  Rams  den's   positive    ocu- 
lar.—  These  eyepieces  do  not 
distort  the  image  as  much  as 
is  the  case  with  the   Huyghe- 
nian    oculars,    and    they    are,, 
therefore,    more    suitable    for 
micrometer     measurements. 
They    consist    of    two    plano- 
convex lenses,  with  the  convex 
surface    turned    toward    each 
other. 

3.  Compensating  oculars,  — 
The    eyepieces   by  this,  name 

are  manufactured 
by  Zeiss,  and  are 
adjusted  for  use 
with  the  apochro- 

rnatic  objectives.     Two  classes  are  made  :  — 
<     CQ        a.    Searching   oculars,  which    are   used   for 
reducing  to   the   lowest   possible   degree   the 
magnification  produced  by  the  objective  and  facilitating   the 
location  of  the  image. 


OCULARS  AND   OBJECTIVES  23 

b.  Working  eyepieces.  These  are  also  the  results  of  the 
inventive  genius  of  Zeiss,  and  were  designed  especially  to  be 
used  in  conjunction  with  the  apochromatic  objectives  made  by 
the  same  house.  They  are  numbered  i,  2,  4,  6,  8,  12,  and  18. 
"  The  number  which  denotes  how  many  times"  an  eyepiece,  when 
used  at  a  given  tube-length,  increases  the  initial  magnifying 
power  of  the  objective  affords  the  measure  of  the  eyepiece 
magnification  and,  at  the  same  time,  furnishes  the  figures  for 
their  rational  numeration.  On  this  basis  the  series  of  our 
compensating  eye-pieces  is  arranged  according  to  their  magnify- 
ing powers.  The  magnification  obtained  by  combining  a  com- 
pensating eyepiece  with  any  apochromatic  objective  is  found  by 
multiplying  its  number  by  the  initial  magnification  of  the  objec- 
tive. An  objective  of  3.0  mm.  focus,  for  example,  yields  in  itself 
a  magnification  of  83.3  (calculated  for  the  conventional  distance 
of  vision  of  250  mm.) ;  eyepiece  12  therefore  gives  with  this  ob- 
jective a  magnification  of  12  x  83. 3  =  1000  diameters."  (ZEISS.) 

These  compensating  oculars  are  corrected  for  aberration  in  the 
extra-axial  portions  of  the  objectives.  It  has  been  well  known 
that  all  objectives  of  considerable  aperture  produce  color  rays  on 
the  margin  of  the  visual  field,  even  though  the  central  rays  may 
come  through  the  lens  free  from  confusion.  This  difficulty  has 
been  overcome  by  the  use  of  oculars  which  have  been  made  to 
possess  an  equivalent  error  but  of  the  opposite  character,  or  in 
other  words  the  image  formed  by  the  red  -ray  is  larger  than  that 
formed  by  the  blue,  and  in  this  manner  the  eyepieces  are  made 
to  correct  the  unequal  magnification  of  different  colors,  and  the 
resulting  image  is  free  from  all  color  and  confusion. 

"  The  mounts  of  the  eyepieces  are  such  that  the  lower  focal 
points  of  all  the  numbers  of  each  series  lie  in  the  same  plane 
when  the  eyepieces  are  inserted  in  the  body-tube.  No  altera- 
tion of  focus  is  therefore  required  on  changing  the  eyepiece, 
and  the  optical  tube,  i.e.  the  distance  between  the  upper  focal  point 
of  the  objective  and  the  lower  of  the  eyepiece,  which  is  the  deter- 
mining element  of  the  magnifying  power,  remains  constant.  In 
the  Continental  microscopes  this  tube-length  is  180  mm.,  irrespec- 


BIOLOGICAL  LABORATORY  METHODS 


tive  of  small  differences  between  the  various  objectives,  the  length 
of  the  body,  from  the  screw-collar  of  the  objective  to  the  upper 
of  the  tube  on  which  the  eyepieces  rest,  being  160  mm. 

"  The  numbering  of  these  eyepieces  is  carried  out  on  the  plan 
proposed  by  Professor  Abbe.  Accordingly,  the  number  which 
denotes  how  many  times  an  eyepiece,  when  used  with  a  given 
tube-length,  increases  the  initial  magnifying  power  of  the  objec- 
tive, affords  a  correct  measure  of  the  eyepiece  magnification,  and 
at  the  same  time  furnishes  the  figures  for  their  rational  numera- 
tion. In  this  way  our  compensating  eyepieces  are  numbered  ac- 
cording to  their  magnifying  powers,  viz.:  2,  4,  6,  8,  12,  18,  27. 

"  The  magnification  obtained  by  the  combination  of  a  com- 
pensating eyepiece  with  any  apochromatic  objective  is  obtained 
by  multiplying  its  number  by  the  initial  magnification  of  the 
objective  as  given  in  the  following  table :  — 


Numerical 
Aperture 

Equivalent  focus 
in  mm. 

Initial 
Magnification 

Dry 

Series 

0.30 

24.0 

1  6.0 

10.5 
15-5 

0.65 

12.0 

8.0 

21 
31 

o-95 

6.0 
4.0 
3-0 

42 
63 
83 

Water  Immersion 

1.25 

2-5 

IOO 

Homogeneous 
Immersion 

1.30 

2.0 

83 

125 
I67 

1.40 

3-° 

2.O 

83 
125 

OCULARS  AND   OBJECTIVES  2$ 

An  objective  of  3.0  mm.  focus,  for  example,  yields  in  itself  a 
magnification  of  83.3  (calculated  for  the  conventional  distance 
of  vision  tof  250  mm.);  eyepiece  12  in  conjunction  with  this 
objective  gives  therefore  a  magnification  of  12x83.3  =  1000 
diameters."  (ZEISS.) 

The  Bausch  &  Lomb  Optical  Company  of  this  country  have 
followed  the  Zeiss  Company  in  making  a  set  of  compensating 
oculars  to  be  used  with  their  series  of  apochromatic  objectives. 
These  oculars,  like  the  Zeiss  pattern,  have  chromatic  error 
opposite  to  that  of  the  objective,  and  these  two  lenses  acting  on 
each  other  eliminate  the  chromatic  error  and  produce  images 
which  are  clear  and  free  from  color.  The  oculars  are  numbered 
2,  4,  8,  12,  1 6.  The  number  2  is  used  as  a  finder  and  for  pho- 
tography ;  4  and  8  are  for  general  work ;  1 2  is  called  into  use 
when  the  utmost  power  of  the  objective  is  required;  and  16  is 
intended  for  aid  in  securing  the  finest  adjustment  in  the  objective. 

The  magnification  can  be  obtained  by  "  dividing  250  mm., 
the  distance  for  distinct  vision,  by  the  focal  length  of  the 
objective  and  multiplying  the  quotient  by  the  number  of  the 
ocular  ;  thus  the  lowest  power  of  the  series  is  equal  to  (250  mm. 
-f-  38)  x  2  =13,  and  the  highest  to  (250  mm.  -^3-5)x  16  = 
1136."  The  tube-length  used  with  these  objectives  and  com- 
pensating oculars  is  the  standard  length  of  160  mm. 

4.  Projection  oculars. — These  eyepieces  are  used  for  project- 
ing an  image  on  a  screen  or  for  photographic  purposes  in  making 
photo-micrographs.  They  are  corrected  so  that  they  may  be 
used  with  the  apochromatic  objectives,  and  the  magnification  is 
determined  in  the  same  manner  as  in  the  case  of  the  compen- 
sating oculars.  The  magnification  of  the  image  on  the  screen, 
or  ground  glass  of  the  camera,  may  be  determined  by  dividing 
the  distance  between  the  ocular  and  the  image  by  the  focal 
length  of  the  objective  and  multiplying  the  result  by  the  num- 
ber stamped  on  the  ocular.  The  distance  must  be  expressed 
in  millimeters,  because  the  other  factors  of  the  equation  are  so 
expressed.  This  will  hold  good  for  long  distances,  but  for 
short  spaces  the  results  are  not  so  accurate. 


26 


BIOLOGICAL  LABORATORY  METHODS 


The  field  is  limited  by  the  diaphragm  between  the  two  systems 
of  lenses.  The  projection  lens  is  so  mounted  that  it  may  be 
moved  from  or  toward  the  diaphragm.  Before  beginning  pho- 
tographic operations,  it  is  best  to  focus  on  the  screen,  or  ground 
glass,  by  extending  or  pushing  into  the  tube  the  projection-glass 
until  the  outlines  of  the  diaphragm  are  distinctly  marked,  and 


Fig.  17 

Sectional  and  longitudinal  view 

of  the  Projection  Eye-pieces. 

O/i  full  size.) 


Plan  view. 


then  the  apparatus  is  adjusted  for  work.  If  the  microscope  is 
near  the  screen,  it  will  be  necessary  to  extend  the  projection- 
glass  from  the  diaphragm. 

In  the  use  of  the  projection  ocular  care  must  be  exercised  in 
adapting  it  to  the  objectives.  The  oculars  made  by  Zeiss  are 
numbered  2,  4,  3,  and  6.  The  2  and  4  systems  are  adjusted 
for  the  Continental  tubes,  or  160  mm.,  and  they  have  an  ampli- 
fication of  2  and  4  times.  The  3  and  6  oculars  are  to  be  used 
with  the  English  tube  of  250  mm.  They  are  specially  designed 
for  the  apochromatic  objectives  and  should  only  be  used  with 
them  when  results  of  the  highest  order  are  required.  In  their 
use  also  care  should  be  exercised  that  the  length  of  the  tube  is 
fixed  at  that  figure  for  which  the  oculars  are  manufactured. 


OCULARS  AND   OBJECTIVES 


The  images  produced  by  these  eyepieces  are  remarkably 
clear  and  accurate,  and  the  photographs  made  by  them  are 
exact  reproductions  of  the  images  seen  by  the 
observer  through  the  ordinary  ocular. 

5.  Micrometer  ocular.  —  This  is  an  eyepiece 
made  according  to  the  Huyghenian  pattern 
with  the  eye-lens  adjustable  so  that  a  focus 
may  be  secured  on  the  measurements  of  the 
micrometer  resting  on  the  diaphragm  between 
the  eye-lens  and  the  field-glass.  Figure  14  gives 
a  correct  idea  of  this  ocular.  The  micrometer  that  accompanies 
this  instrument  is  shown  in  Fig.  18,  which  consists  of  a  disk  of 


FIG.  18.  —  Ocular 
Micrometer. 


FIG.  19.  —  Filar  Micrometer. 

glass  divided  into  arbitrary  lines  which,  when  compared  with  the 
stage  micrometer,  will  give  the  size  of  the  image  produced  by  the 
object.     It  is  best  to  construct  a  table  for  each 
objective,  with  the  assistance  of  the  stage  microm- 
eter, so  that  the  values  of  the  divisions  on  the 
eyepiece  micrometer  may  be  quickly  determined. 
Field  of  Filar          ^'    Filar  micrometer.  —  This    instrument   con- 
Micrometer,       sists  of  an  ocular  with  cross-hairs  so  arranged  that 


28        '  BIOLOGICAL  LABORATORY  METHODS 

they  can  be  moved  by  a  micrometer  screw  across  the  field  of 
observation.  The  milled-head  screw  on  the  left  controls  one  of 
the  vertical  hairs.  The  graduations  on  the  circumference  on 
the  right  of  the  figure  give  the  value  of  the  motion  of  the  microm- 
eter screw ;  the  reading  is  to  0.005  mm-  The  horizontal  and 
one  vertical  hair  are  stationary,  while  the  other  vertical  hair  is 
moved  back  and  forth  across  the  field.  To  measure  with  this 
instrument  a  stage  micrometer  must  be  used  in  connection  with  it. 
If  one  of  the  compensating  oculars  is  used  with  this  microm- 
eter in  connection  with  the  apochromatic  objectives,  it  will 
not  be  necessary  to  construct  the  table  of  magnification,  since 
the  enlargement  can  be  read  off  at  once  without  calculation, 
because  the  divisions  in  the  ocular  have  been  carefully  estimated 
with  a  known  tube-length  and  with  each  apochromatic  objective, 
so  that  each  division  on  the  micrometer  represents  as  many 
micra  (o.ooi  mm.  /x1)  as  there  are  millimeters  in  the  focal 
length  of  the  objective.  For  instance,  if  the  Zeiss  series  of 
objectives  and  ocular  6  are  used,  the  magnification  will  be  as 
follows :  — 

Apochromatic  objectives  16  mm.  8  mm.  4  mm.  3  mm.  2.5  mm.  2  mm.  1.5  mm. 
Value  of  intervals  16  /*       8 /x       4  /*       3  /*       2.5^       2  /*         1.5/4 

Thickness  of  cover-glasses.  —  With  the  use  of  the  high-power 
objectives  it  becomes  absolutely  necessary  that  the  thickness  of 
the  cover-glass,  which  protects  the  section,  should  be  known. 
This  is  essentially  important  because  of  the  refraction  of  the 
light  produced  by  the  cover-glass,  and  the  influence  the  glass 
has  over  the  residue  of  chromatic  and  spherical  aberration  found 
in  nearly  all  objectives.  Although  this  influence  is  inappreciable 
in  the  low-power  objectives,  still,  when  the  immersion  lenses  are 
used,  this  factor  becomes  very  important,  and  the  thickness  of 
the  cover-glass  must  be  known  and  the  correction  allowed  for  it 
in  the  adjustment  of  the  objective  and  in  the  length  of  the  tube. 

iThe  unit  of  measure  is  called  "  micron  "  —  which  is  .001  of  a  millimeter.  This 
measure  was  first  suggested  in  1859  by  Harting,  and  in  1869  Listing  proposed  that 
the  Greek  letter  M  be  used  to  designate  the  .001.  And  16  /u.  would  therefore  mean 
.001  X  16  or  .016  millimeter,  or  16  microns. 


OCULARS  AND   OBJECTIVES  29 

Several  excellent  instruments  have  been  made  by  manu- 
facturers for  measuring  the  thickness  of  cover-glasses,  and  the 
two  illustrated  in  Figs.  20  and  21  give  first-class*  results. 
Figure  20  is  made  by  the  Bausch  &  Lomb  Optical  Company,  and 
is  used  by  placing  the  cover-glass  in  the  opening  between  the 


FIG.  20.  —  Cover-glass  Gauge.     (B.  &  L.) 

screws  passing  through  the  axis  of  the  instrument.  This  screw 
is  termed  the  micrometer-screw.  By  turning  the  milled  head  at 
the  right  of  the  instrument  the  two  screws  are  brought  into  con- 
tact with  the  glass,  and  the  thickness  is  read  off  under  the  knife- 
edge  on  top  of  the  cylinder.  The  divisions  are  in  thousandths  of 
an  inch  and  in  hundredths  of  a  millimeter.  The  scale  also  gives 
the  exact  tube-length  to  be  used  for  each  cover-glass  measured. 


30  BIOLOGICAL   LABORATORY   METHODS 

Figure  21  is  manufactured  by  the  Zeiss  Company,  and  the 
measurement  is  effected  by  pushing  the  lever  to  one  side,  insert- 
ing the  cover-glass  between  the  jaws  of  the  clip  projecting  from 
one  side  of  the  gauge,  and  then  reading  the  thickness  in  milli- 
meters or  inches  by 
means  of  the  pointer 
on  the, disk. 

The  objective.  — 
This  is  the  most  im- 
portant part  of  the 
microscope;  the 
material  of  which  it 
is  made  must  be  of 

the    best,    and    its 
FIG.  21.  —  Cover-glass  Gauge.     (Zeiss.)  , 

manufacture      must 

be  by  the  most  skilful  opticians.  Great  skill  has  been  developed 
in  America  and  abroad  in  calculating  the  lenses  for  objectives, 
and  workmen  of  remarkable  expertness  have  been  trained  in 
their  manufacture. 

In  the  selection,  therefore,  of  a  microscope  the  student  with 
limited  means  should  purchase  a  good  stand  with  few,  if  any, 
accessories  on  the  stage,  and  put  most  of  his  money  in  one  or 
two  of  the  best  objectives,  of  comparative  low  power,  to  be  found 
in  the  market.  As  his  work  advances  in  interest,  and  his 
income  will  permit,  he  may  add  gradually  to  his  list  of  objectives, 
so  that  in  the  course  of  time  he  will  possess  an  outfit  with  which 
he  will  be  able  to  execute  first-class  work  of  a  most  delicate 
character. 

Objectives  for  compound  microscopes  are  all  achromatic. 
That  is  to  say,  they  are  so  constructed  that  the  colored  rays 
decomposed  out  of  the  white  light  by  the  first  lens  are  recomposed 
by  the  next  lens  into  white  light  and  the  image  is  produced  clear 
and  sharp.  When  the  three  principal  rays  of  the  spectrum  are 
united  by  the  lens  system  of  the  objective  in  one  point  of  the 
axis  and  the  correction  of  two  different  colors  has  been  at 
the  same  time  accomplished,  such  an  objective  is  called 


OCULARS   AND   OBJECTIVES 


apochromatic.  These  are  now  manufactured  in  Germany  and  in 
America,  and  they  are  the  finest,  most  expensive,  and  most 
satisfactory  objectives  to  be  obtained  in  the  world.  The  image 
made  by  them  is  distinct  and  sharp  to  the  outer  limits  of  the 
field,  and  the  results  seem  to  be  most  perfect  in  all  respects. 
Objectives  are  generally  divided  into  two  classes:  — 

1 .  Dry  objectives,  and 

2.  Immersion  objectives. 

The  first,  or  dry  series,  are  used  for  examining  objects  in  air ; 
that  is  to  say,  there  is  no  liquid  interposed  between  the  lens  and 
the  slide. 

The  second  class  requires  the  interposition  of  a  drop  of  water 
or  oil  between  the  lens  and  the  slide-cover,  to  catch  those  rays 


ft  (Dry.)  |  (Dry.)  &  (Oil.) 

FlG.  22. —  Dry  and  Oil  Immersion  Objectives.     (B.  &  L.) 

of  light  which  are  considerably  refracted  by  the  cover-glass,  and 
which,  without  the  aid  of  the  liquid,  would  pass  beyond  the 
reach  of  the  objective. 

The  immersion  objectives  constructed  for  oil  are  called 
homogeneous.  Cedar  oil  is  usually  found  best  adapted  for  these 
glasses.  This  oil  has  the  same  index  of  refraction  as  crown 
glass,  viz. :  1.515.  The  index  of  refraction  of  water  (1.336)  being 
lower  than  oil,  some  of  the  rays  are  not  caught  by  it,  and  hence 
it  is  not  so  useful  as  an  immersion  medium.  The  homogeneous 


32  BIOLOGICAL  LABORATORY   METHODS 

objectives  have  a  greater  frontal  distance  and  give  clearer  images 
than  are  possible  with  dry  objectives. 

In  the  manufacture  of  objectives  by  different  houses  in  this 
country  and  abroad,  the  shape,  size,  and  screw-threads  of  the 
lens-mounts  were  almost  as  numerous  in  variety  as  was 
the  number  of  manufacturers.  But  as  the  demand  for  delicate 
and  multiplied  work  with  the  microscope  increased  in  different 


Slide 


FIG.  23.  —  Section  of  Oil  Immersion  Objective  showing  Position  of  Oil. 

parts  of  the  world,  came  also  the  demand,  on  the  part  of  the 
microscopists,  for  greater  facilities  in  handling  the  instrument. 
It  was  then  that  the  manufacturers  realized  the  importance  of 
making  certain  parts  of  the  instrument,  made  at  one  factory, 
interchangeable  with  similar  portions  made  by  another  firm. 

The  most  important  part  of  the  microscope  subjected  to  this 
evolution  was  the  screw-thread  used  in  attaching  the  objective 
to  the  tube  of  the  instrument.  Even  in  this  late  day  we  have 
the  German  thread,  the  English  thread,  and  the  American  thread. 
Some  years  since,  however,  the  Royal  Microscopical  Society  of 
England  devised  a  screw  which  is  now  called  "The  Society's 
Screw"  and  most  of  the  opticians  in  America  and  abroad  have 
adopted  it,  so  that  now  stands  purchased  in  one  country  may 
receive  objectives  made  in  another  portion  of  the  world. 

Objectives  were  formerly  designated  by  letters,  but  the  almost 
universal  custom  now  is  to  mark  them  with  numbers  indicating 


OCULARS  AND   OBJECTIVES  33 

the  focal  distance  of  the  lens.  And  it  would  also  be  of  great  ad- 
vantage if  all  objectives  contained  figures  to  designate  the  angular 
aperture,  i.e.  the  angle  formed  by  the  two  most  divergent  rays  of 
light  emanating  from  the  axial  point  of  the  object  and  entering 
the  objective.  Professor  Abbe  found  that  it  was  difficult  to  satis- 
factorily compare  all  objectives  by  the  use  of  the  angular  aper- 
ture, since  some  lenses  were  dry,  some  water,  and  others  were 
homogeneous  or  oil-immersion  objectives  ;  and  it  was  found  neces- 
sary, therefore,  to  take  into  consideration  the  refractive  index 
of  the  medium  intervening  between  the  objective  and  the  cover- 
glass,  so  he  introduced  in  1873  the  method  which  he  termed 

Numerical  aperture  (N.A.).  —  This  represents  the  capacity  of 
the  lens  for  receiving  the  rays  of  light  from  the  object  and  trans- 
mitting them  to  the  image.  "  The  ratio  between  the  radius  of 
the  emerging  pencils  measured  in  the  upper  focal  plane  of  the 
equivalent  focal  length  of  the  latter."  We  may  express  this 
ratio  by  the  following  formula  :  — 

N.A.  =  n  sin  «, 

in  which  n  designates  the  index  of  refraction  of  the  medium 
between  the  object  or  cover-glass,  and  sin  u  is  the  sine  of  half 
the  angle  of  aperture. 

By  means  of  this  formula  we  can  now  accurately  compare  an 
objective  of  one  class  with  that  of  another  class.  For  example, 
if  we  take  a  dry  objective,  the  medium  of  which  is  air,  and  apply 
it  to  our  formula,  we  will  get  i  for  the  value  of  n,  by  the  use  of  a 
table  of  natural  sines  ;  for  the  immersion  lenses  the  value  of 
n  will  vary  according  to  the  character  of  the  media  used  between 
the  objective  and  the  cover-glass,  governed  of  course  by  the 
value  of  the  refractive  index  of  the  medium  used  in  each  case. 
To  illustrate  the  use  of  this  formula,  let  us  take  one  example  in 
the  case  of  the  oil  immersion  objective.  The  refractive  index 
of  cedar  oil  is  1.52,  the  angle  of  aperture  of  the  objective  is,  say, 
60°,  half  this  angle  is  30°.  From  the  table  of  natural  sines  we 
get  for  30°  0.50  ;  applying  to  the  formula  we  have 

N.A.  =  1.52  x  0.50  =  0.76. 


34 


BIOLOGICAL   LABORATORY  METHODS 


APERTURE  TABLE 

[Copied  from  Journal  of  the  Royal  Microscopical  Society} 


Numerical 
Aperture 
(«  sin  u  =  a) 

Correspond'g  Angle  (zu)  for 

Limit  of  Resolving  Power  in 
Lines  to  an  Inch 

| 

JP 

Pene- 
trating 
Power 

(0 

.V? 

^  II 

^ 

<u   ro 

s| 

Homogeneous 
Immersion 
(««=!.  5«) 

White  Light 
(A=o.526g  /a, 
Line  E) 

Monochro- 
matic 
(Blue)  Light 

(A—  0.4861  /!x, 

Line  F) 

Photog- 
raphy 
(A=o.4ooo  p., 
Near  Line  h) 

•52 
•5i 
•50 
•49 
.48 

•47 
.46 

•45 
•44 
•43 
.42 
.41 
.40 
•39 
•38 
•37 
•36 
•35 
•34 
•33 
•32 
•30 
.28 
.26 
.24 

.22 
.20 
.18 
.16 
.14 
.12 
.IO 
.08 
.06 
.04 
.02 
.OO 
0.98 
0.96 
0-94 

— 

— 

I  80°    0' 

i66°5i 
i6i°23 

157°  12 

153°  39 
1  50°  32 
147°  42 

145°    6 
142°  39 

140°  22 
I38°  12 

136°    8 
134°  10 
132°  16 
1  30°  26 
1  28°  40 
126°  58 
125°  18 
1  23°  40 

122°     6 

1  20°  33 

1  17°  35 
1  14°  44 
iii°  59 

109°  20 

io6°45 
104°  15 
101°  50 
99°  29 
97°  1  1 
94°  55 
92°  43 
90°  34 
88°  27 
86°  21 
84°  18 
82°  1  7 
80°  17 
78°  20 
76°  24 

146,543 

J45,579 

144,615 
143,651 
142,687 

HI,723 
140,759 

139,795 
138,830 
137,866 
136,902 
135,938 

1  34,974 
134,010 
133,046 
132,082 
131,118 

130,154 
129,189 
128,225 
127,261 
125,333 
123,405 
121,477 
119,548 
117,620 
115,692 
113,764 
in,835 
109,907 
107,979 
106,051 
104,123 
102,195 
100,266 
98,338 
96,410 
94,482 

92,554 
90,625 

158,845 
157,800 

I56,755 
i55,7io 
154,665 
153,620 

152,575 
15^530 
150,485 
149,440 

H8,395 
!47,350 
146,305 
145,260 
144,215 

143,17° 
142,125 
141,080 
140,035 
138,989 
137,944 
135,854 
!33,764 
1  3  1,  674 
129,584 
127,494 
125,404 

123,314 
121,224 

H9,i34 
117,044 

H4,954 
112,864 
110,774 
108,684 
106,593 
104,503 
102,413 
100,323 
98,223 

193,037 
191,767 
190,497 
189,227 

'87,957 
186,687 

185,417 
184,147 
182,877 
181,607 
1  80,337 
179,067 

177,797 
176,527 

I75>257 
i73,9»7 
172,717 

I7M47 
170,177 
168,907 

167,637 
165,097 
162,557 
160,017 
'57,477 
!54,937 
152,397 
149,857 

147,317 
144,777 
142,237 
139,698 

i37,I58 
134,618 
132,078 
129,538 
126,998 
124,458 
121,918 
"9,378 

2.310 

2.280 
2.250 

2.  22O 
2.190 

2.161 

2.132 

2.103 

2.074 
2.045 

2.016 

1.988 

1.960 

1.932 
1.904 

1.877 

1.850 
1.823 
1.796 

1.769 
1.742 
1.690 
1.638 
1.588 
1-538 
1.488 
1.440 
1.392 
1.346 
1.300 
1.254 

I.2IO 
I.I66 
I.I24 
1.082 
I.O4O 
I.OOO 

.960 

.922 
.884 

.658 

.662 
.667 
.671 
.676 
.680 

.685 

.690 

.694 

.694 
.709 
.709 
.714 
.719 

•725 
.729 

•735 
•741 
•746 
•752 
.758 
•769 
.781 

•794 
.806 
.820 

•833 

.847 
.862 
.877 
•893 
.909 
.926 

•943 
.962 
.980 

I.OOO 
I.O2O 

1.042 
1.064 

— 

— 

— 

— 

— 

— 

I  80°     0' 

165°  56' 
155°  38' 
148°  42' 

142°  39' 
137°  36' 
133°   4' 
128°  55' 

125°    3' 

121°  26' 

118°  o' 
1  14°  44' 
in0  36' 
1  08°  36' 
105°  42' 
102°  53' 

100°  10' 

97°  3i' 
94°  56' 
92°  24' 

89°  56' 

— 

1  80°     0' 
I57°     2' 
I47°  29' 

140°    6' 

OCULARS  AND   OBJECTIVES 


35 


APERTURE  TABLE    (Continued) 
[Copied  from  Journal  of  the  Royal  Microscopical  Society} 


Numerical 
Aperture 
(«  sin  u=a) 

Correspond'g  Angle  (2»)  for 

Limit  of  Resolving  Power  in 
Lines  to  an  Inch 

| 

rt  w^-s 

p 

Pene- 
trating 
Power 

0) 

b| 

*l 

V3 

*   ro 

*i 

Homogeneous 
Immersion 

(«=1.52) 

White  Light 
(A=o.5269/u, 
Line  E) 

Monochro- 
matic 
(Blue)  Light 
(A=o.486ij«., 
Line  F) 

Photog- 
raphy 
(A.=o.4ooo/u., 
NearLfiie-6) 

O.Q2 

133°  51' 

87°  32' 

74°  30 

88,697 

96,143 

116,838 

.846 

1.087 

o.go 

1  28°  1  9' 

85°  10' 

72°  36 

86,769 

94,053 

114,298 

.810 

I.  Ill 

0.88 

1  23°  1  7' 

82°  51' 

70°  44 

84,841 

91,963 

IH,758 

•774 

1.136 

0.86 

1  1  8°  38' 

80°  34' 

68°  54 

82,913 

89,873 

109,218 

.740 

1.163 

0.84 

1  14°  1  7' 

78°  20' 

67°    6 

80,984 

87,783 

106,678 

.706 

1.190 

0.82 

I  10°  10' 

76°    8' 

65°  18 

79,056 

85,693 

104,138 

.672 

I.22O 

0.80 

1  06°  1  6' 

73°  58' 

63°  3i 

77,128 

83,603 

101,598 

.640 

1.250 

0.78 

102°  31' 

7i°  49' 

6i°45 

75,200 

81,513 

99,058 

.608 

1.282 

0.76 

98°  56' 

69°  42' 

60°   o 

73,272 

79,423 

96,518 

.578 

I.3I6 

0.74 

95°  28' 

67°  37' 

58°  16 

7I»343 

77,333 

93,979 

.548 

I-35I 

0.72 

92°   6' 

65°  32' 

56°  32 

69,415 

75,242 

9i,439 

.518 

1.389 

0.70 

88°  51' 

63°  31' 

54°  50 

67,487 

73,152 

88,899 

.490 

1.429 

0.68 

85°  41' 

6i°3o' 

53°   9 

65,559 

71,062 

86,359 

.462 

I.47I 

0.66 

82°  36' 

59°  30' 

51°  28 

63,631 

68,972 

83,819 

436 

I.5I5 

0.64 

79°  36' 

57°  3i' 

49°  48 

61,702 

66,882 

81,279 

.410 

1.562 

0.62 

76°  38' 

55°  34' 

48°    9 

59,774 

64,792 

78,739 

.384 

I.6I3 

0.60 

73°  44' 

53°  38' 

46°  30 

57,846 

62,702 

76,199 

.360 

1.667 

0.58 

70°  54' 

51°  42' 

44°  5  1 

55,9i8 

60,612 

73,659 

.336 

1.724 

0.56 

68°    6' 

49°  48' 

43°  H 

53,990 

58,522 

71,119 

•3H 

1.786 

0-54 

65°  22' 

47°  54' 

4i°  37 

52,061 

56,432 

68,579 

.292 

1.852 

0.52 

62°  40' 

46°     2' 

40°   o 

5°,J33 

54,342 

66,039 

.270 

1.923 

0.50 

60°    o' 

44°  10' 

38°  24 

48,205 

52,252 

63,499 

.250 

2.000 

0-45 

53°  30' 

39°  33' 

34°  27 

43,385 

47,026 

57>!49 

.203 

2.222 

0.40 

47°   9' 

35°   o' 

30°  3i 

38,564 

41,801 

50,799 

.160 

2.500 

0-35 

40°  58' 

30°  30' 

26°  38 

33,744 

36,576 

44,449 

.123 

2.857 

0.30 

34°  56' 

26°    4' 

22°  46 

28,923 

3I,35I 

38,099 

.090 

3-333 

0.25 

28°  58' 

21°  40' 

i8°56 

24,103 

7.6,126 

31,749  . 

.063 

4.000 

0.20 

23°   4' 

17°  18' 

15°    7 

19,282 

20,901 

25,400 

.040 

5.OOO 

0.15 

1  7°  14' 

12°  58' 

11°  19 

14,462 

15,676 

19,050 

.023 

6.667 

O.  IO 

11°  29' 

8°  38' 

7°  34 

9,641 

10,450 

12,700 

.010 

10.000 

0.05 

5°  44' 

4°i8' 

3°  46 

4,821 

5,252 

6,350 

.003 

20.000 

Expressed  in  another  way  we  may  say  that  the  dry  objective, 
where  air  is  the  medium  between  the  objective  and  the  cover- 
glass,  catches  a  smaller  per  cent  of  light  rays  than  the  objective 
with  water  interposed  in  the  place  of  air ;  or  if  we  assume  a 
maximum  theoretical  aperture  of  180°  for  the  dry  objective,  no 


36         BIOLOGICAL  LABORATORY  METHODS 

more  light  will  pass  through  it  from  the  object  than  through  the 
water  objective  with  an  aperture  of  97°,  or  through  an  oil  immer- 
sion lens  of  82°  aperture. 

The  preceding  table,  copied  from  the  pages  of  the  Journal  of 
the  Royal  Microscopical  Society,  is  given  to  enable  the  student 
to  compare  his  objectives  with  each  other  with  but  little  trouble. 
This  table  was  made  out  by  means  of  the  formula  given  above. 
"  The  numerical  aperture  of  a  lens  determines  all  its  essential 
qualities.  The  brightness  of  the  image  increases  with  a  given 
magnification,  other  things  being  equal,  as  the  square  of  the 
aperture  ;  the  revolving  and  defining  powers  are  directly  propor- 
tional to  it ;  the  focal  depth  varies  inversely  as  the  square  and 
so  forth."  (ABBE.) 

From  what  has  been  said  in  the  previous  pages  we  may 
classify  objectives  not  only  into  dry  and  immersion,  but,  when 
we  take  into  consideration  the  remarkable  evolution  which  has 
taken  place  within  recent  years  in  the  development  of  these 
important  parts  of  the  microscope  in  overcoming  chromatism 
and  spherical  aberration,  we  may  regroup  them  into  two  distinct 
systems  called :  — 

1.  Achromatic  objectives. 

2.  Apochromatic  objectives. 

The  achromatic  objectives  comprise  the  great  majority  of 
those  used  on  better  grades  of  microscopes  until  the  discovery 
of  the  Jena  glass.  They  give  distinctness  of  image,  but  limited 
to  one  color  of  the  light  rays,  the  green-yellow ;  the  other  rays 
give  confusion.  The  color  correction  is  also  limited  to  only  one 
zone  of  the  objective,  and  only  two  colors  are  united  in  that 
zone.  It  is  therefore  possible  for  the  various  colored  images  to 
fall  on  the  same  spot  in  pairs,  but  there  is  a  considerable  varia- 
tion in  the  focus  of  each  pair  of  colors.  This  result  is  accom- 
plished by  the  combination  of  concave  and  convex  lenses  made 
of  different  kinds  of  glass  (see  page  6).  The  best  of  these  ob- 
jectives are  also  aplanatic. 

The   apochromatic  objectives  were  devised  by  Abbe,  of  the 


OCULARS  AND   OBJECTIVES 


37 


Zeiss  Optical  Company,  and  they  give  marked  improvements 
over  the  older  series  or  the  achromatic.  They  are  made  to 
focus  three  colors  in  the  same  point,  and  color  correction  is  uni- 
form in  all  zones.  "  The  amount  of  focal  differences  of  the 
various  sections  of  the  spectrum,  from  the  visible  to  far  into 
the  chemically  active  portion,  are  reduced  to  fractions  varying 
from  ^  to  -^  of  their  original  magnitude,  i.e.  they  are  practically 
eliminated,  and  this  is  done  in  equal  degree  for  all  zones  of  the 
objective.  The  images  due  to  each  single  color,  thus  individu- 
ally corrected,  are  rendered  perfectly  coincident  and  collectively 
form  the  final  image." 

With  these  objectives  it  makes  no  difference  what  may  be  the 
character  of  light  which  is  emitted  from  the  object ;  the  quality 
of  the  image  is  not  marred,  but  remains  sharp  and  clear,  and 
the  natural  colors  of  the  object  are  faithfully  reproduced,  even 
to  the  most  delicate  tints. 

There  are  other  names  given  by  opticians  to  objectives  manu- 
factured by  them,  but  all  the  best  objectives  in  the  market  will 
naturally  fall  under  one  or  the  other  of 
the  two  classes  given  above. 

Dr.  H.  van  Heurck  thus  summarizes 
the  chief  forms  of  modern  apochromats 
(Ann.  Soc.  Beige  de  Micr.  XXIII,  1899, 
pp.  43-73.  Also  Jour.  Roy.  Mic.  Soc.,  p. 
115,  1900):  — 

i.  Objective  16  mm.,  N.A.  0.30  (Fig. 
24).  —  The  system  consists  of  three 
lenses :  the  frontal  plano-convex  of  low 
curvature ;  the  median  double ;  and  the 
superior  slightly  convex,  and  is  in  reality 
formed  of  a  highly  curved  biconvex  lens 
between  two  menisci.  The  low  numerical 


FIG.  24.  — Objective  16 
mm.,  N.A.  0.30. 


aperture  of  this  objective  permits  of  employment  only  for  histo- 
logical  studies,  for  which  it  gives  very  beautiful  and  delicate 
images. 

2.    Objective  8  mm.,  N.A.  0.65   (Fig.  25). —  This  is  a  quad- 


38         BIOLOGICAL  LABORATORY  METHODS 

ruple  combination  of  seven  simple  lenses  of  different  kinds  of 
glass,  and  of  different  curves.  It  is  a  very  successful  objective, 
and  if  a  microscopist  possesses  only  one  apochromat,  he  ought 
to  select  this.  With  different  compensating  oculars  the  mag- 
nification varies  from  62  to  562  diameters.  It  shows  Nobert's 
sixth  group  well  with  axial  illumination,  and  resolves  the  striae 
of  Pleurosigma  angulatum. 

3.    Objective  6  mm.,  N.A.  0.95  (Fig.  26).  —  The  construction 
of  this  objective  resembles   that   of   the   last,  but  the   curves 


FIG.  25.  — Objective  8  mm., 
N.A.  0.65. 


FIG.  26.  —  Objective  6  mm. 
N.A.  0.95. 


are  sharper,  especially  that  of  the  frontal  lens.  It  will  revolve, 
with  axial  light,  Nobert's  twelfth  group  (2830  lines  to  the  milli- 
meter). 

4.  Objective    4    mm.,   N.A.    0.95 ;    and  j   mm.,  N.A.   0.95 
(Fig.  27).  —  These  resemble  the  last  in  design.     The  3  mm.  will 
resolve  Nobert's  thirteenth  group,  and  it  is  somewhat  superior 
to  4  mm. 

5.  Objective    2.5    mm.,   N.A.    i.    25,    water-immersion    (Fig. 
28).  —  Construction    like    last ;    a    very    beautiful     objective. 


OCULARS  AND   OBJ] 


39 


OF  THE     '       X 

VNJVERSITY  ) 

OF 

££i^i£^ 

A  slight  alteration  of  the  diaphragm  produces  a  marked 
effect  on  the  image,  and  with  axial  light  the  resolution  lies 
between  Nobert's  thirteenth  and  fourteenth  groups.  With 
electric  oblique  light  Nobert's  eighteenth  group  is  distinctly 
resolved. 

6.  Objective  3  mm.,  N.A.  1.40,  homogeneous  (Fig.  29).  —  This 
contains  ten  lenses.  The  simple  frontal  lens  slightly  exceeds 
a  hemisphere,  and  the  object  of  such  a  lens  is  to  produce  a 


FIG.  27.  —  Objective 
4  mm.,  N.A.  0.95. 


FIG.  28.  —  Objective 
2.5  mm.,  N.A.  1.25. 


FIG.  29. — Objective  3  mm., 

N  A     T  AC, 


j 

N.A.  1.40. 


notable  amplification  of  the  object  without  introducing  at  the 
same  time  chromatic  and  spherical  aberrations.  It  reduces 
the  pencil  aperture  from  1.40  to  0.65.  The  first  doublet 
discharges  similar  functions ;  at  first  it  diminishes  the  pencil 
divergence,  and  afterward  intentionally  introduces  a  certain 
amount  of  chromatic  and  spherical  aberration  for  more  com- 
plete correction  of  the  upper  lenses.  The  first  triplet  destroys 
spherical  and  chromatic  aberration,  and  the  second  triplet 
the  secondary  spectrum. 


4o 


BIOLOGICAL  LABORATORY   METHODS 


LIST  OF  THE  APOCHROMATIC  OBJECTIVES 

(ZEISS) 


Numerical  Aperture 

Equivalent  Focus 
in  mm. 

Initial 
Magnification 

Dry  Series 

0.30 

24.0  * 
1  6.0 

10.5 

'5-5 

0.65 

I2.01 
8.0 

21 
31 

o-95 

6.0  1 

4.0 

3-° 

42 
63 
83 

Water  Immersion 

1.25 

2-5 

100 

Homogeneous        I 
Immersion 

I 

1.30 

3.0 

2.O 

i-5 

83 

"5 

167 

1.40 

3-° 

2.O 

83 
125 

TABLE  OF  MAGNIFICATIONS  OF  THE  APOCHROMATIC   OBJEC- 
TIVES  WITH   THE   COMPENSATING   EYEPIECES 
Calculated  for  an  image  distance  of  250  mm.  =  10  in. 
(ZEiss) 


S         Vi 

Focus  of  the 

Eyepiece 

Working  Eyepieces 

Objective 

2 

4 

6 

8 

12 

18 

27 

24.0 

21 

42 



83 

I25 

187 

281 

16.0 

31 

62 

94 

I25 

187 

281 

— 

12.  0 

42 

83 

167 

250 

375 

562 

8.0 

62 

125 

187 

250 

375 

562 

6.0 

83 

167 

333 

500 

75° 

112 

4.0 

'25 

250 

372 

500 

•750 

1125 

— 

3-o 

I67 

333 

498 

667 

IOOO 

1500 

— 

2-5 

200 

400 

600 

800 

I2OO 

1800 

— 

2.O 

250 

500 

750 

IOOO 

1500 

2250 

— 

1-5 

333 

667 

IOOO 

1334 

2000 

3000 

— 

1  The  three  objectives  24  mm.,  12  mm.,  and  6  mm.  of  the  dry  series  are  con- 
structed exclusively  for  the  lo-inch  tube,  all  the  others  are  adjusted  either  for  the 
Continental  or  English  tube. 


OCULARS  AND   OBJECTIVES  41 

The  practical  value  of  the  magnifications  obtained  with  the  high-power 
eyepieces  in  conjunction  with  lenses  of  relatively  short  focus  is  found  by  mul- 
tiplying its  number  by  the  initial  magnification  of  the  objective.  An  objective 
of  3.0  mrn.  focus,  for  example,  yields  in  itself  a  magnification  of  83.3  (calculated 
for  the  conventional  distance  of  vision  of  250  mm.);  eyepiece  12  in  conjunc- 
tion with  this  objective  gives  therefore  a  magnification  of  12  X  83.3  =  1000 
diameters.  (ZEiss.) 

It  will  be  of  some  importance  to  the  student  to  know,  before 
making  a  purchase,  how  many  objectives  are  required  to  enable 
him  to  perform  his  work  with  satisfaction  and  accuracy.  Ex- 
perience has  demonstrated  that  the  complete  outfit  for  all  inves- 
tigations, from  the  ordinary  to  the  most  delicate  work,  must 
consist  of  the  following  objectives  :  — 

2  inches  (50  mm.), 
i  inch  (25  mm.). 
1-2  inch  (12.5  mm.). 
1-4  inch  (6.5  mm.), 
i-io  inch  (2.6  mm.). 

In  this  series  4.2  and  2.0  mm.  may  replace  the  6.5  and  2.6 
mm.  The  2.6  and  2.0  mm.  objectives  should  be  of  the  im- 
mersion type.  The  apochromatic  lens,  as  has  been  stated,  is 
the  best,  and  it  will  be  the  part  of  wisdom  to  purchase  this  kind 
of  objective. 

For  the  use  of  students  who  are  not  far  advanced  in  histology, 
the  following  objectives  will  be  found  sufficient :  — 

i  inch  (25  mm.). 

1-5  or  1-6  inch  (5  mm.  or  4.2  mm.). 

How  to  use  the  microscope.  —  A  delicate  piece  of  apparatus  in 
good  condition  is  a  most  excellent  assistant  to  the  scientific 
worker ;  but  if  it  is  spoiled  by  careless  handling,  it  will  be  a 
source  of  trouble  ever  afterward.  The  microscope  when 
properly  treated  will  be  a  faithful  servant  for  many  years,  and 
will  greatly  aid  the  microscopist  in  solving  many  interesting 
problems  which  will  be  beyond  his  reach  if  the  glasses  and 
delicate  parts  have  been  previously  injured  by  bad  management. 


42  BIOLOGICAL   LABORATORY   METHODS 

Therefore  if  the  student  desires  to  become  a  successful  investi- 
gator in  Nature's  laboratory,  he  must  learn  a  few  rules  in  rela- 
tion to  the  use  and  care  of  the  apparatus  and  diligently  endeavor 
to  apply  them. 

1.  Never  let  the  hands  come  in  contact  with  any  glass  sur- 
faces, particularly  the  lenses  in  the  objectives  and  the  oculars. 

2.  Keep  everything  about  the  instrument  clean  ;  eschew  dust 
as  one  would  the  pestilence.     If   the    lenses  require  cleaning, 
exercise  the  greatest  care  and  gentleness  hi  rubbing  them,  and 
use  the  softest  old  linen,  or,  better  still,   the  Japanese   paper, 
which  is  free  of   particles  of  grit.     Do  not  permit  alcohol  to 
touch  the  metal  portions  of  the  microscope,  because  it  will  dis- 
solve the  lacquer  with  which  the  metal  is  covered.     In  order  to 
locate  particles  of  dust  on  the  lenses,  twist   the   tube  of  the 
microscope  while  the  eye  is  looking  through  the  ocular,  and  with 
the   other  hand   hold   the   ocular  stationary.     If  the  particles 
revolve  with  the  tube,  it  is  a  sure  evidence  that  the  dust  is  on 
the  objective,  and,  vice  versa,  on  the  ocular. 

3.  If  the  tubes  or  other  portions  of  the  instruments  need  oiling, 
moisten  a  clean  cloth  with  the  best  watchmaker's  oil  and  rub 
the  parts.     Take  care  that  none  of  the  oil  reaches  the  lenses. 

4.  While  using  chemical  reagents  it  will  be  wise  to  protect  all 
metallic  apparatus  from  fumes  of  acids. 

5.  Never  focus  downward,  for  fear  of  injuring  the  objective 
lens  or  breaking  the  slide  containing  the  object.     Always  focus 
up.     In   order  to  do  this  work  correctly,  bring  the   objective 
down  by  means  of  the  rack  and  pinion  movement,  with  the  eye 
on  a  level  with  and  looking  across  the  stage ;  then  it  will  be 
possible  to  lower  the  objective  near  the  slide  without  bringing 
the  two  in  contact.     Now,  with  the  eye    looking   through  the 
ocular,  focus  up  with  the  coarse  adjustment  until  the  image  is 
brought  into  view  and  finish  with  the  fine  adjustment. 

6.  Begin  work  with  the  low  powers  of  the  microscope  until 
an  objective  is  found  which  will  show  the  image  of  object  dis- 
tinctly in  all  its  details.     There  is  nothing  to  be  gained  in  using 
a  high  power  when  a  low  power  will  do. 


OCULARS  AND   OBJECTIVES  43 

7.  In  using  the  immersion  objectives,  be  certain  to  clean  the 
lenses  of  all  oil  when  the  work  is  completed  and  before  putting 
them  away. 

8.  Chlorate  of  lime  is  an  excellent  absorbent  of  water,  and 
when  placed  in  the  microscope  case  it  will  dry  the  atmosphere 
and  thus  prevent  the  rusting  of  the  steel  portions  of  the  instru- 
ment. 

9.  The  laquered  parts  of  the  instrument  should  be  wiped  dry 
and  clean  after  the  work  is  completed  for  the  day,  because  the 
touch  of  the  perspiring  fingers  will  produce  an  indelible  stain  if 
left  for  several  days. 

10.  If  by  accident  the  objective  lens  should  come  in  contact 
with  soft  balsam  or  other  resinous  substances,  remove  the  objec- 
tive from  the  microscope  at  once  and  wipe  off  the  gum  with  a 
soft  cloth  or  with  Japanese  paper,  moistened  in  benzol ;  dry  at 
once  with  a  fresh  piece  of  paper  before  the  solvent  enters  the 
mounting  and  dissolves  the  cement  holding  the  lens  in  place. 

Care  of  the  eye.  —  There  is  so  much  danger  that  the  student 
will  seriously  injure  his  eyes  when  he  first  begins  to  use  the 
microscope,  the  teacher  should  take  every  precaution  to  prevent 
the  trouble,  and  he  should  lay  special  stress  upon  the  importance 
of  properly  adjusting  the  instrument  and  himself  to  the  light. 
The  field  of  the  microscope  should  be  well,  but  not  brilliantly, 
illuminated ;  and  direct  sunlight  must  not  be  used  for  ordinary 
observation.  The  proper  application  of  the  diaphragm  is  impor- 
tant here. 

The  best  light  is  secured  from  the  sky  or  from  a  cloud.  The 
observer  will  be  in  the  best  position  for  work  when  he  sits  fac- 
ing the  window,  because  then  his  hands  will  not  be  in  the  way 
of  the  illumination  while  he  is  manipulating  the  stage  and  its 
appliances.  In  this  position  the  light  may  be  painful  to  the 
eyes,  at  times,  but  this  trouble  can  be  obviated  by  placing  a 
screen  made  of  cardboard  on  the  table  in  front  of  the  observer. 
The  lower  edge  of  this  screen  should  be  raised  above  the  table 
high  enough  to  permit  the  light  from  the  window  to  reach  the 
reflector  and  stage  of  the  microscope. 


44         BIOLOGICAL  LABORATORY  METHODS 

As  soon  as  the  eyes  begin  to  pain  or  show  indications  of 
fatigue,  the  work  with  the  microscope  must  be  suspended  and 
other  duties  should  be  assigned  the  student  in  order  that  the 
eyes  may  rest  and  recover  their  strength. 

Use  of  the  diaphragms.  —  These  are  disks,  with  different  cen- 
tral openings,  which  are  placed  just  below  the  stage  and  as  near 
the  object  as  possible.  They  are  used  for  cutting  off  all  rays  of 
light  reflected  from  the  mirror  except  those  which  are  illuminating 
the  object.  The  correct  use  of  the  diaphragm  must  be  determined 
by  experiment,  and  its  best  effects  are  only  secured  by  experi- 
ence. As  a  general  rule  it  may  be  stated  that  the  diaphragm, 
when  used  without  the  condenser,  must  be  well  opened  for  low 
powers  which'  have  low  apertures.  When  used  with  the  con- 
denser the  object  is  to  narrow  down  the  pencil  of  light  coming 
from  the  condenser,  and  in  this  case  the  diaphragm  should  be 
small  for  low  powers  and  large  for  high  powers. 

Dark  ground  illumination  is  obtained  by  adapting  a  diaphragm 
with  the  opening  in  the  shape  of  a  ring  and  the  centre  oblique. 
This  form  of  opening  permits  of  oblique  lighting  and  renders 
the  field  dark  while  the  image  stands  out  bright  and  striking. 
Dark  ground  illumination  is  also  secured  by  making  the  dia- 
phragm eccentric,  that  is,  by  moving  it  to.  one  side. 

On  many  of  the  microscopes  now  manufactured  the  iris  dia- 
phragm is  placed,  and  this  form  enables  the  observer  to  increase 
or  diminish  the  size  of  the  opening  at  pleasure  and  with  but 
little  trouble.  The  term  "  stopped  down  "  is  generally  used  to 
designate  the  action  of  the  diaphragm. 


CHAPTER   III 

APPARATUS    AND   ACCESSORIES 

BESIDES  the  microscope  mentioned  in  preceding  chapters,  the 
laboratory  should  also  contain  the  following  accessories  :  — 

The  microtome.  —  There  are  several  first-class  instruments 
for  cutting  sections  sold  by  some  of  the  leading  manufacturers, 
and,  since  they  are  all  made  on  the  same  general  principle,  it 
will  be  only  necessary  to  describe  two  or  three  which  possess 
the  most  convenient  facilities  for  work. 

The  microtome  is  an  instrument  constructed  for  cutting  thin 
sections  of  animal  and  vegetable  tissues,  so  that  a  satisfactory 
examination  of  the  cells  may  be  made  with  the  microscope.  The 
essential  features  of  this  instrument  are  a  keen-edged  knife  firmly 
held  on  a  sliding  carrier,  and  a  clamp  for  holding  the  specimen 
while  the  sections  are  being  cut. 

Before  the  microtome  became  the  necessary  adjunct  to  the 
laboratory,  the  method  of  cutting  sections  was  by  holding  the 
specimen  between  the  thumb  and  forefinger  of  the  left  hand  and 
by  a  dexterous  sweep  of  the  razor  held  in  the  right  hand.  After 
considerable  practice  the  student  was  able  to  secure  sections  of 
remarkable  thinness. 

The  next  step  in  the  development  was  by  Valentine's  knife, 
which  consisted  of  two  blades  parallel  to  each  other  and  sepa- 
rated by  screws.  These  screws  permitted  the  adjustment  of  the 
blades,  so  that  they  could  be  separated  at  small  distances.  This 
knife  was  soon  discarded  by  microscopists  as  inadequate  to  the 
demands. 

The  hand  microtome,  shown  in  Fig.  30,  next  came  in  favor, 
and  is  still  used  in  some  laboratories  where  a  cheap  form  of 
instrument  is  required.  This  microtome  is  so  constructed  that 

45 


46 


BIOLOGICAL  LABORATORY   METHODS 


the  specimen  can  be  clamped  in  the  well  a  by  means  of  the 
screw  b.  The  knife  held  in  the  hand  slides  over  a  glass  plate, 
and  the  feed  at  c  lifts  the  object  through  a  minimum  distance  of 
ten  microns.  In  the  hands  of  skilful  students  this  instrument 
will  yield  satisfactory  results. 

An  improved  form  of  the  hand  microtome  is  shown  in  Fig.  31, 
which  is  constructed  to  clamp  on  the  edge  of  the  work-table  by 
means  of  the  screw  a,  and  the  specimen  is  fastened  in  the  instru- 
ct 


a 


FIG.  30.  —  Hand  Mi- 
crotome. 


FjG.  31.  —  Hand  Microtome  vyith  Clamp 
for  Table. 


ment  by  the  thumb-screw  b.  On  top  of  the  microtome  are  two 
glass  ways  along  which  the  knife  moves,  and  the  milled  head  c 
raises  the  specimen  to  the  plane  of  the  knife.  Sections  can  be 
cut  with  this  microtome  as  thin  as  five  microns.  An  ordinary 
razor  can  be  used  with  this  apparatus. 

No  instrument  belonging  to  the  microscopist's  outfit  has 
undergone  such  marked  changes  as  the  microtome.  It  has 
developed  from  the  simple  hand  instrument  to  a  microtome  of 
great  precision.  There  are  many  different  patterns  on  the 
market,  but  the  entire  number  may  be  classed  under  two  heads, 
differing  in  the  method  by  which  the  object  is  raised  to  the 
plane  of  the  knife  :  — 


APPARATUS   AND   ACCESSORIES 


47 


1.  Microtomes  distinguished  by  having  the  specimen  raised 
to  the  knife  by  means  of  a  micrometer-screw  in  a  vertical  direc- 
tion, or  modifications  of  this  movement.     The  Bausch  &  Lomb 
Optical  Company's  instruments  are  types  of  this  class. 

2.  Microtomes  distinguished  by  raising  the  specimen  to  the 
knife  by  means  of  an  inclined  plane.    The  Thomas's  and  Reichert 
instruments  give  illustrations  of  this  class. 

Bausch  &  Lomb's  microtomes.  —  This  firm  makes  several  styles 
of  microtomes  belonging  to  the  vertical  micrometer-screw  move- 


FiG.  32.  — B.  &  L.  Automatic  Microtome. 


ment.  The  automatic  microtome  (Fig.  32)  is  their  most  elabo- 
rate instrument.  "  The  object  remains  stationary  during  cutting, 
being  held  by  an  extremely  rigid  clamp  adjustable  in  two  planes 
and  vertically.  The  knife  is  carried  on  a  block,  and  is  angular 
in  section,  sliding  in  perfectly  ground  ways  so  constructed  as  to 
give  the  greatest  solidity  with  the  least  friction.  The  motion 
of  the  knife  operates  the  feed-screw,  leaving  the  hand  free  for 
the  manipulation  of  the  sections.  The  feed  is  from  two  to  sixty 


48  BIOLOGICAL  LABORATORY  METHODS 

microns,  in  two  microns.  The  carriage  containing  the  feed 
arrangement  and  object  carrier  is  adjustable  along  the  whole 
front  of  the  microtome,  so  that  any  desired  cutting  angle 
or  length  of  stroke  can  be  had.  The  split  nut  permits  the 
object  holder  to  be  instantly  lowered  to  beginning  position 
when  screw  has  been  fed  out.  An  automatic  irrigator  for  keep- 
ing the  knife  flooded  with  alcohol  or  other  fluid  when  cutting 
celloidin  preparations  consists  of  a  glass  cup  on  an  arm  adjust- 
able over  the  knife.  The  flow  of  liquid  is  regulated  by  a  screw- 
valve. 

Minot's  automatic  rotary  microtome. — This  instrument,  Fig.  33, 
was  designed  by  Dr.  Charles  S.  Minot,  of  Harvard  University 
Medical  School,  and  is  well  and  favorably  known  in  many  labo- 


FiG.  33.  —  Minot's  Rotary  Microtome. 

ratories.  It  is  especially  adapted  for  serial  or  ribbon  sectioning. 
The  object  carrier  is  adjustable  in  three  planes.  The  feed  is  so 
controlled  that  it  will  produce  sections  1,2,  3,  4,  5,  6,  7,  8,  9,  10, 
n,  12,  13,  14,  15,  16,  17,  18,  19,  20,  21,  22,  23,  24,  25  microns  in 
thickness.  The  object  to  be  cut  is  cemented  to  the  disk  shown 
in  the  figure  just  above  the  knife.  There  is  also  an  extra  clamp 
for  holding  paraffin  blocks  which  may  be  used  on  the  machine 


APPARATUS  AND   ACCESSORIES 


49 


instead  of  the  disk.  The  fly-wheel  is  accurately  adjusted,  so 
that  it  moves  with  the  greatest  ease  and  smoothness.  A  ribbon 
carrier,  shown  in  Fig.  34,  accompanies  this  microtome,  and  it  is 
used  for  catching  the  serial  sections.  This  rotary  microtome 
is  adapted  only  for  cutting  paraffin  sections,  and  it  works  more 
rapidly  than  the  precision  instrument.  The  rapid  movement  of 
the  wheel  causes  not  only  the  disk  carrying  the  object  to  approach 


FIG.  34.  —  Ribbon  Carrier. 


the  knife,  but  also  an  up  and  down  rapid  motion  is  given  to  the  disk, 
so  that  the  sections  are  automatically  cut  by  the  simple  turning  of 
the  wheel.  When  the  ribbon  carrier  is  used  in  serial  sectioning, 
the  knife  is  transferred  from  its  position  shown  in  the  microtome 
figure  to  the  slots  a  and  b  on  the  ribbon  carrier,  and  this  latter 
apparatus  is  substituted  for  the  ordinary  knife  holder  shown  in 
the  figure  illustrating  the  microtome.  As  the  sections  leave  the 
blade  of  the  knife,  they  move  out  on  the  ribbon  in  the  order  cut. 
Minot's  automatic  precision  microtome.  —  This  instrument,  Fig. 
35,  is  totally  different  from  the  rotary  microtome  designed  by 
Dr.  Minot,  and  it  is  capable  of  cutting  sections  of  uniform  thick- 
ness of  one  micron.  The  object  holder  is  the  Naples  universal 
clamp,  which  can  be  adjusted  in  two  planes  by  rack  and  pinion. 
The  vertical  motion  of  the  object  carrier  is  accomplished  by 
means  of  a  fine  adjustment  screw  and  triangular  prism  similar 
to  that  used  on  the  microscopes  manufactured  by  the  Bausch 
&  Lomb  Optical  Company.  This  microtome  is  constructed  for 
carrying  the  ribbon  attachment  used  on  the  rotary  machine. 


5O         BIOLOGICAL  LABORATORY  METHODS 

This  ribbon  carrier  is  fastened  to  the  back  of  the  knife,  and  serial 
sections  can  be  made  on  the  microtome  with  ease  and  rapidity. 
The  shape  of  the  knife  is  peculiar,  which  is  clearly  shown  in  the 
illustration.  This  knife  is  made  especially  for  this  instrument. 


FIG.  35.  —  Minot's  Precision  Microtome. 


Reichert's  microtome  with  inclined  plane  and  endless  working 
micrometer-screw.  —  "The  great  advantages  of  the  construction 
of  this  microtome  are  not  merely  that  the  object  holder  works  on 
an  inclined  plane  and  in  a  strong  frame  between  two  swallow- 
tailed  metal  guides,  but  that  the  micrometer-screw,  when  it  has 
worked  out,  can,  by  means  of  a  simple  and  ingenious  arrange- 
ment, be  rotated  through  180°,  so  that  a  kind  of  perpetual  motion 
results.  The  knife  slides  with  a  working  distance  of  28  cm. 
The  knife  slide,  on  three  accurately  worked  projecting  guides,  is 
heavy,  and  is  thoroughly  secured  by  means  of  a  clamp ;  a  slip  of 
the  knife  in  the  cutting  of  hard  objects  is  impossible. 


APPARATUS  AND  ACCESSORIES  5 1 

"  The  object  slide,  7.5  cm.  long,  is  solidly  built,  and  moves  on 
an  inclined  plane  only  12.5  cm.  long,  which  makes  an  angle  of 
15°  with  the  horizon.  It  moves  with  perfect  safety  and  rigidity 
between  two  metal  guides,  and  on  account  of  the  inclination  of 
its  path,  the  horizontal  projection  of  its  movement  is  propor- 
tionately slow.  The  object  holder  is  easily  adjusted  in  three 


FIG.  36.  —  Reichert's  Microtome. 

kirections  of  space,  in  the  vertical  by  rack  and  pinion,  and  can, 
y  the  simple  action  of  a  screw  d,  be  set  higher  or  lower,  or 
even  removed  altogether. 

"  The  micrometer-screw  is  fixed,  that  is,  it  is  never  necessary 
to  remove  it  from  the  mother  screw.  The  fine  adjustment  is  by 
a  micrometer-screw  of  the  construction  usual  with  microtomes, 
a  circular  sheath  with  accurately  divided  tooth  periphery  envelop- 
ing the  screw.  A  circular  segment  ^,  graduated  in  degrees,  can 
be  pushed  into  position,  and  adjusted  so  that  the  number  of 
teeth  (i  tooth  =  i  //,)  can  be  set.  By  moving  the  handle  h,  the 


52  BIOLOGICAL  LABORATORY   METHODS 

upper  end  of  the  circular  segment  strikes  against  a  metal  cross- 
piece,  which  serves  as  a  stop,  and  thus  rotates  the  toothed  wheel, 
and  moves  on  the  micrometer-screw  so  that  uniformly  thick  sec- 
tions are  automatically  obtained.  When  the  micrometer-screw 
is  run  out,  it  can,  in  a  circular  notch  of  the  vertical  guide  of  the 
microtome,  be  rotated  through  180°,  and  then  commence  again, 
the  other  end  of  the  micrometer-screw  being  now  in  contact  with 
the  object  slide.  Thus  the  result  is  an  uninterrupted  working 
of  this  screw.  The  arrangement  is  especially  useful  in  serial- 
section  making."  (Jour.  Roy.  Mic.  Soc.,  p.  59,  October,  1901.) 
How  to  care  for  the  microtome.  —  i .  Keep  the  parts  subject  to 
friction  well  oiled,  so  that  the  carrier  will  move  with  the  least 
difficulty.  Use  watch  oil  or  pure  paraffin  oil  of  25°. 

2.  Do  not  press  on  the  knife  while  cutting,  but  permit  it  to 
move  with  its  carriage  by  pushing  and  not  pressing,  because  other- 
wise the  bearing  surfaces  will  soon  become  worn  and  uneven. 

3.  Keep  the   knife  sharp  as  a  razor,  and  see  that  no  gaps 
occur  on  its  edge. 

How  to  sharpen  the  microtome  knife.  —  For  the  accomplish- 
ment of  first-class  work  the  microtome  knife,  Figs.  37  and  38, 
must  be  in  the  best  possible  condition.  It  must  be  ground  so 


FIG.  37.  —  Microtome  Knife  with  Handle  for  Sharpening. 

keen  that  a  hair  resting  on  its  edge  will  be  severed.  The  same 
degree  of  care  must  be  taken  with  this  knife  in  sharpening  it 
and  in  keeping  it  sharp  that  is  taken  with  the  razor  in  the  hands 
of  a  first-class  barber. 

If  the  knife  is  in  a  dull  condition,  begin  with  the  Belgian  yel- 
low hone,  which  is  an  open-grained  stone,  and  it  will  cut  the 


APPARATUS  AND   ACCESSORIES  53 

metal  to  an  edge  quickly  and  will  take  out  any  gaps  if  such 
happen  to  be  present.  Lather  the  surface  of  this  stone  freely 
with  palm-oil  soap  before  applying  the  knife,  and  keep  the 
surface  well  covered  with  the  lather  during  the  entire  time  of 
sharpening.  This  soap  is  preferable  to  oils  ordinarily  used  for 
this  purpose  because  it  keeps  the  pores  of  the  stone  open  and 
thus  gives  clean  and  free  cutting  surfaces.  After  the  knife  is 
properly  ground  on  the  yellow  stone  so  that  all  gaps  and  in- 
equalities are  taken 
out  of  the  edge,  it 
is  then  ground  on 
the  blue-green 
water  stone,  which 

is    a    harder    and  ^i^  "n*|»>f«»i 

finer-grained  stone 
than       the      vellow  FIG.  38.  —  Knife  mounted  for  the  Microtome. 

Belgian  rock.  On  this  stone  the  knife  is  sharpened  until  a 
fine  thread  is  formed  on  the  edge,  which  may  be  determined 
by  passing  the  edge  across  the  finger  nail ;  when  this  thread 
is  secured,  the  knife  is  passed  over  the  stone  without  press- 
ure until  a  uniform  keenness  is  obtained  along  the  entire  edge, 
which  may  be  determined  by  bringing  the  edge  in  contact 
with  the  moistened  skin  of  the  hand  ;  and  if  the  sensation  is  that 
the  knife  will  enter  the  flesh,  the  grinding  has  been  carried  far 
enough.  Of  course,  this  grinding  must  be  done  in  the  same 
manner  as  that  used  in  honing  a  razor,  i.e.  the  edge  must  always 
be  kept  toward  the  front  as  the  knife  is  passed  back  and  forth 
on  the  stone. 

When  the  proper  edge  is  obtained  on  the  knife  with  the  blue 
stone,  clean  it  thoroughly  with  a  soft  cloth,  taking  care  not  to 
bring  the  cloth  in  contact  with  the  edge,  and  then  strop  it  on 
fine  leather  covered  with  a  prepared  dressing.  Pressure  must 
not  be  great  in  this  stropping,  otherwise  the  delicate  edge 
secured  on  the  blue  hone  will  be  damaged.  Draw  the  knife 
over  the  leather  gently  and  lightly,  and  continue  this  work  until 
a  hair  can  be  cut  freely  along  the  entire  extent  of  the  edge. 


54 


BIOLOGICAL  LABORATORY  METHODS 


Wipe  carefully  with  a  soft  cloth,  and  the  knife  is  then  ready 
for  the  microtome.  At  frequent  intervals  during  cutting  the 
sections,  give  the  knife  a  few  strokes  on  the  strop,  so  that  its 
edge  will  retain  its  keenness. 


FIG.  39.  —  Forceps. 


Forceps. —  These  are  used  for  grasping  the  cover-glasses  and 
holding  small  objects.  The  tips  should  be  grooved  on  the 
inside,  so  that  the  objects  may  not  slip  out.  There  should  be  at 
least  two  or  three  instruments  on  each  student's  desk  —  one 
with  straight  tips  and  the  other  bent  (Fig.  39). 


FIG.  40.  —  Scissors. 


APPARATUS  AND   ACCESSORIES 


55 


Scissors.  —  These  must  be  of  the  very  finest  quality  and  may 
be  either  straight  or  curved,  although  both  will  be  found  con- 
venient (Fig.  40).  The  usual  length  is  n  cm.  The  edges  must 
be  kept  keen  when  fine  work  is  desired. 


FlG.  41.  —  Needle-holders  and  Needles. 

Needle-holders.  —  The  needles  are  for  dissecting  purposes,  and 
the  sizes  generally  used  are  Nos.  n  and  12.  The  holders  (Fig. 
41)  are  so  constructed  that  one  size  of  needle  may  be  substi- 
tuted for  another  by  means  of  a  clamp  at  the  end  of  the  holder. 
Needles  may  be  purchased  from  the  dealers,  containing  straight, 
bent,  and  flattened  points  of  various  shapes. 

Scalpels.  —  Dissection  knives  are  essential  in  microscopic 
work,  and  various  shapes  of  blades  are  now  sold  by  the  manu- 


56  BIOLOGICAL  LABORATORY   METHODS 

facturers.  The  one  illustrated  in  the  cut  (Fig.  42)  is  the  best 
form  for  general  use.  It  has  an  ebony  handle  and  contains  a 
blade  4.4  cm.  long.  The  edge  should  be  as  keen  as  a  razor  at 
all  times ;  and  the  surface  of  the  knife  must  be  pol- 
ished after  each  using,  so  that  rust  may  not  spoil  its 
working  properties.  The  knife  must  be  made  of  the 
finest  English  steel,  well  tempered,  so  that  it  will  hold 
its  edge  under  ordinary  careful  treatment. 

Section  lifters.  —  These  are  made  of  brass,  nickel- 
plated,  1.9  cm.  wide  on  the  spatula  end,  and  13.1  cm. 
in  length.  This  instrument  is  used  for  transferring 
the  sections  from  one  vessel  to  another  without  damag- 
ing their  delicate  structure  (Fig.  43). 

Spring-clips  or  compressors.  —  These  are  made  of 
wire,  nickel-plated,  and  are  used  for  holding  the  cover- 
glass  in  position  over  the  object  while  the  slide  is  being 
cleaned  and  the  mounting  medium  is  hardening  previ- 
ous to  spinning  the  final  finishing  ring. 

Brushes.  —  Camel's-hair  pencils  in  quills  and  mounted 
on  short  handles  will  be  found  useful  in  numerous 
manipulations :  in  transferring  the  delicate  sections 
from  the  microtome  knife  to  the  watch-glasses ;  in 
moistening  the  surface  of  the  knife  while  the  sections 
are  being  cut ;  in  dusting  off  cover-glasses  and  slips  ; 
in  spinning  rings  of  gum  on  the  slips  to  seal  the  cover- 
glass.  The  pencils  should  be  in  assorted  sizes,  from 
Nos.  i  to  8. 

Bell-glasses.  —  These  are  convenient  for  preserving 
the  slides  from  dust  during  the  process  of  mounting. 
These  glasses  should  be  from  10  to  30  cm.  in  diameter. 
FScai4p2eT  A   number  of  bell-glasses  on  the  work-table  will   be 

found  very  useful. 

Glass  benches.  — While  the  mounts  are  undergoing  the  various 
processes  it  becomes  necessary  to  set  them  aside  under  certain 
conditions,  and  in  a  laboratory  where  many  slides  are  being 
made,  the  space  on  the  table  must  be  economized.  These 


APPARATUS  AND  ACCESSORIES 


57 


benches,  therefore,  when  piled  one  on  top  of  the  other,  and 
covered  with  a  bell-glass  (Fig.  44),  will  hold  a  greater  number 
of  slides  than  could  be  possibly  stored  under  the  bell- 
glass  without  them.  The  usual  length  is 
130  mm.,  and  55  mm.  wide. 


FIG.  43.  — Section  Lifters. 

When  these  benches  are  used  the  bell-glass  should  be  procured 
of  sufficient  dimensions  to  accommodate  them. 

Petri  dishes.  —  These  com- 
prise two  glass  vessels  (Fig.  45), 
the  larger  inverted  on  the 
smaller,  and  they  are  used  in 
inoculation  experiments  in  the 
FIG.  44.  —  Glass  Benches.  study  of  bacteria. 


58  BIOLOGICAL   LABORATORY   METHODS 

A   small   wash-bottle.  —  This   apparatus,    so   necessary  in   a 
chemical  laboratory,  will  be  found  useful  on  the  microscopist's 
table  for  holding  distilled  water  and  dilute 
alcohol. 

Watch-glasses. — There  are  several  shapes 
of  watch-glasses  sold  in  the  market,  but 
the  author  has  used  with  satisfaction  a  pat- 
tern of  watch-glass,  called  "  Syracuse  solid 


FIG.  45.  —  Petri  Dishes. 


watch-glass  "  (Figs.  46  and  47),  which  possesses  certain  proper- 
ties of  special  merit.  The  edges  and  bottom  are  accurately 
ground,  so  that  when  one  glass  is  placed  on  top  of  another,  air- 
tight covers  are  thus  provided  for  each  vessel.  This  form  will, 


FIG.  46.  —  Syracuse  Watch- 
glasses. 


FlG.  47.  —  Section  of  Syracuse 
Watch-glasses. 


therefore,  take  up  less  room  on  the  table,  which  is  quite  a  desid- 
eratum. The  bevelled-ground  edges  form  writing  surfaces  on 
which  data  concerning  contents  may  be  written  with  a  pencil. 

Glass  pipettes,  or  dropping  tubes.  —  These  tubes  (Fig.  48)  are 
made  with  one  end  drawn  to  a  point,  and  on  the  other  end  is  a 
rubber  bulb.  They  are  useful  for  transferring  a  drop  of  fluid 


FlG.  48.  —  Pipette,  with  Rubber  Bulb. 


APPARATUS  AND   ACCESSORIES 


59 


Erlenmeyer  Flask. 


Koch  Flask. 


FIG.  49. 


to  the  places  desired  in  the  manipulations.     By  pressing  the 

bulb  and  immersing  the  point  in  the  liquid  and  then  releasing 

the  pressure,  the   fluid  will 

rise   in    the    tube   and   may 

be  transported  with  perfect 

safety.      By    another    slight 

pressure  a  drop  or  more  may 

be  exuded  when  desired. 
Erlenmeyer's    and    Koch's 

flasks.  —  These  vessels  (Fig. 

49),  in  various  sizes,  will  be 

found  serviceable  in  the  labo- 
ratory.   A  supply  of  beakers 

will    also    be    required    for 

numerous  purposes. 

Mortars.  —  Mortars  made  of  iron,  wedgwood,  and  glass  should 

be  placed  in  the  laboratory  for  general  use  in  the  preparation  of 

staining  and  mount- 
ing media. 

Thomas's  dehy- 
drating apparatus.  — 
This,  or  a  similar 
contrivance  (Fig.  50), 
will  be  found  useful 
in  the  preparation  of 
the  sections  while 
staining,  hardening, 
or  dehydrating.  The 
Thomas  apparatus 
consists  of  a  museum 
jar  20  X  21  cm.  con- 
taining a  diaphragm 
of  perforated  hard 
rubber  suspended  76 
mm.  from  the  top. 
Through  the  holes. 


FIG.  50.  —Thomas's  Dehydrating  Apparatus. 


60         BIOLOGICAL  LABORATORY  METHODS 

of  various  sizes,  in  this  diaphragm  are  passed  the  dehydrat- 
ing tubes,  which  are  1.8,2.5,  an^  3  cm-  m  diameter,  and  in 
which  are  placed  the  sections  to  be  treated.  On  the  lower 
ends  of  these  tubes  are  fastened  chamois  skins.  If  dehydration 
is  to  be  accomplished,  the  tubes  are  rilled  with  50  per  cent 
alcohol,  and  in  the  jar  is  placed  95  per  cent  alcohol.  By 
osmotic  action  the  two  will  become  the  same  strength,  and  the 
tissues  can  then  be  treated  with  absolute  alcohol,  if  complete 
dehydration  is  desired.  By  this  method  of  treatment  the 
tissues  do  not  shrink,  since  the  changes  are  not  sudden.  An 
addition  of  calcium  chloride  to  the  jar's  contents  absorbs  the 
water,  and  the  quality  of  the  alcohol  is  not  depreciated.  The 
rapidity  of  the  dehydrating  may  be  increased  or  diminished  by 
the  degree  of  thickness  of  the  chamois  skin  on  the  tubes. 

Turn-table.  —  This  is  one  of  the  absolute  essentials  to  each 
work-table.      There   are   several   patterns   sold   by  dealers   in 


FIG.  51.  —  Turn-table. 


microscopic  apparatus,  differing  from  each  other  in  minor  details, 
but  the  form  illustrated  in  the  cut  (Fig.  51)  contains  all  the 
important  principles  of  the  turn-table.  This  is  the  American 
table,  and  for  all  ordinary  purposes  it  subserves  the  ends 
desired.  Messrs.  Bausch  &  Lomb  have  devised  a  table  with  a 
movable  hand-rest,  which  projects  partly  over  the  circle  of  the 
table  but  not  in  contact  with  it,  thus  giving  a  support  for  the 
hand,  which  is  very  useful  in  those  manipulations  where 
steadiness  of  brush  is  necessary  to  produce  neat  and  accurate 
work.  Glass  slips  can  be'  quickly  centred  on  this  table  by  means 


APPARATUS  AND   ACCESSORIES 


61 


of  adjusting-pins,  and  the  usual  spring-clips  are  also  present  to 
hold  the  clip  firmly  to  the  table.  The  turn-table  is  used  for 
spinning  the  cement  rings  on  the  slide  to  seal  the  mounts  and 
exclude  air  from  the  specimen. 

Water-baths.  —  There  are  so  many  things  for  which  a  water- 
bath  is  required  in  a  well-equipped  laboratory  that  it  is  deemed  best 
to  describe  several  kinds  of  apparatus  adapted  to  this  purpose. 

The  essential  principle  upon  which  these 
baths  are  built  is  a  mass  of  water  contained 
between  an  outer  and  an  inner  cop- 
per vessel.      Heat  applied  to  the 
outer  vessel   causes   the   water   to 
boil  and  thus  transmit  a  known  de- 
gree of  heat  to  smaller  ves- 
sels placed  on  the  bath  or 
in  its  oven. 

If    the    funds    of    the 
laboratory   will    permit 
of  the  expense,  it  will 
be  best  to  provide  each 
desk  with  one  of  these 
water-baths ; 
but    if     the 
outlay      of 
money      is 
an  important 
item  and  the 
director     of 

the  laboratory  has  not  sufficient  funds  to  furnish  each  student 
with  a  water-bath,  then  he  should  purchase  one  or  more  of  the 
larger  sizes  which  contain  compartments  enough  for  the  class. 

The  bath  devised  by  Dr.  W.  S.  Miller  of  the  University  of 
Wisconsin  (Fig.  52)  is  among  the  best  of  its  kind  for  individual 
use.  It  consists  of  a  rectangular,  polished  copper  vessel  with  a 
sheet-iron  base,  to  prevent  the  burning  out  of  the  copper.  The 
top  of  the  bath  contains  the  following  apparatus  :  — 


FIG.  52.  —  Miller's  Water-bath. 


62 


BIOLOGICAL   LABORATORY   METHODS 


1.  Two  cups,  one  57  mm.  in  diameter  and  the  same  in  depth, 
and  the  other  63.5  mm.  in  diameter,  shallow,  and  shaped  some- 
what like  a  watch-glass. 

2.  Five  bottles,  to  be  used   in  various   preparations  of  the 
imbedding  material.     As  will  be  noticed  in  the  cut,  the  bottles 
each  occupy  a  receptacle  sunk  in  the  bath  so  that  uniform  heat 
may  be  secured. 

3.  Two  drawers,  or  trays,  in  which  the  slides  may  be  placed 
to  dry  and  harden  the  gums. 

4.  'A  shelf  upon  which  the  forceps,  needles,  and  other  instru- 
ments used  about  the  bath  may  be  placed  and  kept  at  the  same 

temperature   as  that   of 
the  paraffin. 

5.  There  is  an  open- 
ing at   one  end  of   the 
bath  in  which  the  Bun- 
sen  burner  is  placed. 

6.  A  thermometer  to 
keep     the     temperature 
uniform. 

Another  bath  of  value 
for  individual  use,  de- 
vised by  Dr.  Reeves,  is 
illustrated  in  Fig.  53. 
It  consists  of  heavy 
polished  copper,  with 
paraffin  cup  and  stirrer  and  thermometer.  The  vessel  is 
216  mm.  high  and  254  mm.  wide. 

For  general  use  in  the  laboratory  a  water-bath  something  like 
the  Lillie  pattern  (Fig.  54)  may  be  adopted  with  advantage. 
This  apparatus  was  devised  by  Dr.  F.  R.  Lillie,  of  the  Univer- 
sity of  Chicago.  The  illustration  gives  a  clear  conception  of  this 
water-bath.  It  consists  of  two,  four,  and  six  rows  of  drawers  in 
a  large  chamber.  Each  of  the  drawers  is  25  cm.  long,  10  cm. 
wide,  and  8  cm.  deep,  and  they  are  provided  with  partitions, 
and  are  numbered.  The  ends  and  bottoms  are  made  of  copper, 


FIG.  53.  — Reeves's  Water-bath. 


APPARATUS  AND   ACCESSORIES  63 

and  the  sides  of  perforated  zinc.  There  are  no  partitions 
between  these  drawers,  so  that  the  heat  may  circulate  through 
the  perforated  sides  and  throughout  the  entire  large  chamber. 


FIG.  54.  — Lillie's  Water-bath. 

In  these  partitioned  drawers  may  be  placed  various  vessels  used 
in  infiltrating,  imbedding,  or  any  other  manipulation  requiring 
heat  for  its  proper  completion.  The  large  chamber  consists  of 
an  inner  and  an  outer  vessel,  between  which  the  water  is  placed. 


64 


BIOLOGICAL    LABORATORY   METHODS 


The  outer  vessel  is  made  of  a  non-conducting  material,  so  that 
an  economy  of  heat  is  secured.  A  gas  burner  underneath  fur- 
nishes the  heat  for 
raising  the  tempera- 
ture of  the  water, 
and  a  thermometer 
and  a  thermo-regula- 
tor  are  provided  in 
tubulations,  so  that 
the  degree  of  heat 

FIG.  55. -Paraffin  Imbedding  Box.  "^   be    Under    COn- 

trol. 

Paraffin  imbedding  box.  —  Where  imbedding  is  required  the 
apparatus  shown  in  Fig.  55  is  most  convenient.  It  consists 
of  two  triangular  pieces  of  type  metal,  about  20  mm.  high,  with 
a  glass  plate  to  form  the 
base.  To  use  the  instru- 
ment, wet  the  glass  plate 
with  glycerin  and  gently 
warm  it;  now  place  the 
box  on  the  plate,  ad- 
justed to  the  dimensions 
desired,  and  pour  in  the 
paraffin.  Before  the 
paraffin  is  '  cooled  the 
specimen  is  immersed 
in  it  in  the  position  de- 
sired; and  after  the 
preparation  becomes 
hard,  it  may  then  be 
taken  from  the  metal  box 
and  transferred  to  the 
microtome  to  be  cut  into 
sections. 

Paper    imbedding   box. 
—  This   method   is   con- 


FIG.  56.  —  Paper  Imbedding  Box. 


APPARATUS  AND   ACCESSORIES  65 

venient  in  some  instances  where  the  metal  boxes  are  not  avail- 
able. 

A  piece  of  stiff  paper,  the  size  desired  and  in  the  proportions 
shown  in  Fig.  56,  is  bent  in  the  same  directions  along  the 
lines  AC,  BD,  EF,  GH'y  and  in  opposite  directions  along  the 
lines  12,  3 4,  56,  78.  Now  fold  the  box  in  shape  and  turn  over 
the  ends  along  the  lines  13  and  57,  so  that  these  laps  will  bind 
the  box  and  thus  prevent  collapsing. 


CHAPTER   IV 

PREPARATION    OF   THE   TISSUE    FOR   MOUNTING 

IN  preparing  tissue  for  permanent  preservation,  some  definite 
order  must  be  pursued  or  the  results  will  not  be  satisfactory. 
The  tissue  must  be  cut.  into  very  thin  sections,  so  that  the  cellu- 
lar structure  can  be  examined  by  means  of  transmitted  light; 
but  before  these  sections  can  be  made,  the  specimen  must  be 
subjected  to  certain  treatment  preliminary  to  the  cutting.  In 
some  instances  the  specimen,  if  plant  or  animal,  must  be  killed 
quickly,  so  that  the  cells  will  not  lose  their  normal  condition  ; 
certain  fluids  and  other  compounds  must  be  extracted  from  the 
specimen,  in  order  that  the  chemicals  used  in  the  processes  may 
not  be  interfered  with  in  their  action  on  the  tissue  ;  if  the  speci- 
men is  delicate  and  its  tissue  will  be  crushed  in  the  jaws  of  the 
microtome,  it  will  become  necessary  to  strengthen  the  cell  walls 
with  paraffin  or  celloidin  before  the  cutting  can  be  successfully 
accomplished ;  in  some  cases  the  entire  specimen  must  be 
stained  before  the  cutting  is  done,  and  in  those  instances  special 
treatment  becomes  necessary  before  the  staining  chemicals  will 
penetrate. 

The  general  steps  for  preparing  the  specimens  for  mounting 
and  converting  the  fresh  tissue  into  the  completed  slide  for  per- 
manent preservation  may  be  outlined  as  follows  :  — 

I.  When  Paraffin  is  used:  2.  When  Celloidin  is  used: 

Fixing  and  hardening  the  fresh  tissue.  Fixing  and  hardening  the  fresh  tissue. 

Dehydrating.  Dehydrating. 

Clarifying.  Treatment  with  ether-alcohol. 

Infiltrating.  Infiltrating. 

Imbedding.  Imbedding. 

Cutting  the  sections.  Hardening  with  chloroform. 

66 


PREPARATION  OF  THE  TISSUE  FOR  MOUNTING 


67 


I .   When  Paraffin  is  used  : 

Fixing  the  sections  to  the  slip  and 

removing  imbedding"  material. 
Gaining. 

Vashirig   extra   stain   away  with  al- 
cohol. 

larifying  with  carbol-turpentine,  or 
carbol-xylene. 

>ansfer  to  slip  in  Canada  balsam. 
Place  on  cover-glass. 
)in  finishing  ring. 
sL 


2.   When  Celloidin  is  used : 

Clarifying  with  castor-xylene. 

Cutting  the  sections. 

Fixing  the  sections  to  the  slip  and 
removing  imbedding  material. 

Staining. 

Washing  extra  stain  away  with  al- 
cohol. 

Clarifying  with  carbol-turpentine,  or 
carbol-xylene. 

Transfer  to  slip  in  Canada  balsam. 

Place  on  cover-glass. 

Spin  finishing  ring. 

Label. 


Dr.  Piersol  in  the  Appendix  to  his  Normal  Histology  gives 
e  following  method  for  preparing  specimens  for  imbedding  in 
iraffin,  cutting,  and  mounting  in  Canada  balsam :  — 

1.  Fixation  of  fresh  tissue  in  large  quantity  by  Mtiller's 
luid ;  renewal  when  turbid ;  tissue  remains  2-3  weeks. 

2.  Thorough  washing  in  running  water  2-5  hours.* 

3.  Transfer  to  70  per  cent  alcohol ;  keep  in  dark  ;  change 
ilcohol  whenever  it  becomes    deeply  tinged,  until   it   remains 

)lorless. 

4.  Stain  in  excess  of  borax-carmine,  24-48  hours. 

5.  Transfer  directly,  without  washing,  from  stain  to  acid 
Icohol,  24-48  hours. 

6.  Wa:sh  well  in  70  per  cent  alcohol,  several  times  renewed, 
14  hours. 

"  7.    Transfer  to  80  per  cent  alcohol,  24  hours. 

"  8.    Transfer  to  95  per  cent  alcohol,  24-48  hours. 

"9.    Dehydrate  in  absolute  alcohol,  24-48  hours. 

10.    Transfer   to  pure   chloroform    until    tissue   sinks,   6-8 
mrs. 

"  ii.  Transfer  to  saturated  solution  of  paraffin  in  chloroform, 
hours. 

"  1 2 .  Transfer  to  pure  melted  paraffin,  kept  in  constant  temper- 
ture  of  about  50°  C.,  until  all  chloroform  is  driven  off  6-8  hours. 


68  BIOLOGICAL   LABORATORY   METHODS 

"  13.  Transfer  to  fresh  melted  pure  paraffin  of  consistence 
for  imbedding,  10-15  minutes. 

"14.    Imbed  tissue  in  mould  ;  cool 'rapidly. 

"15.  Section  in  microtome,  first  suitably  trimming  block  for 
cutting. 

"  1 6.    Fix  sections  to  slides  by  gum  or  collodion  clove  oil. 

"17.    Remove  paraffin  by  benzole,  succeeded  by  turpentine. 

"  18.  Drain  off  excess  of  turpentine,  apply  balsam,  and 
cover. 

"  19.    Place  freshly  mounted  slide  in  horizontal  position. 

"  20.  Clean  up  and  permanently  label  when  thoroughly  dry ; 
store  in  suitable  cabinet. 

"  While  the  duration  of  the  several  manipulations  as  indicated 
in  the  above  summary  represents  the  time  usually  required  by 
ordinary  objects,  yet  the  individual  character  of  the  tissue  must 
be  considered  in  each  case,  as  density  exerts  much  influence  on 
the  rapidity  with  which  the  fluids  penetrate. 

"  When  it  is  desirable  to  stain  the  tissue  after  sections  have 
been  cut,  the  above  manipulations  must  be  modified  ;  steps  4,  5, 
and  6  in  such  case  are  omitted,  and  the  tissue  is  at  once  dehy- 
drated. Removal  of  the  paraffin  from  the  fixed  sections  of  the 
slides  (17)  by  benzole,  is  at  once  succeeded  by  the  following 
manipulations :  — 

"  (a)  Transfer  to  95  per  cent  alcohol  to  remove  benzol,  5-10 
minutes. 

"  (ft)  Transfer  to  clean  95  per  cent  alcohol  to  insure  complete 
absence  of  benzol,  5  minutes. 

"  (tf)    Transfer  to  80  per  cent  alcohol,  5  jninutes. 

"  (d)   Transfer  to  70  per  cent  alcohol,  5  minutes. 

"  (e)    Stain  in  borax-carmine  solution,  10-15  minutes. 

"  (/)  Differentiate  in  acid  alcohol  (10  per  cent),  6-10  minutes. 

"  (g)   Wash  in  70  per  cent  alcohol  (renewed),  10-15  minutes. 

"  (h)   Transfer  to  80  per  cent  alcohol,  15  minutes. 

"  (t)    Transfer  to  95  per  cent  alcohol,  15  minutes. 

"  (/)    Dehydrate  thoroughly  in  absolute  alcohol,  15  minutes. 

"  (k)    Clear  sections  in  oil  of  turpentine,  5  minutes. 


PREPARATION  OF  THE  TISSUE  FOR   MOUNTING 


69 


"  (/)    Mount  in  balsam  as  indicated  above  in  18. 

"When  hsematoxylin  is  used  as  the  stain,  the  steps  ^, 
are  omitted,  and  replaced  by  — 

"  (ee)    Transfer  to  distilled  water,  5  minutes. 

"  (ff)    Stain  in  properly  diluted  haematoxylin  fluid  until  suffi- 
ciently dark,  8-10  minutes. 

"  (gg)  Wash  well  in  distilled  water  to  remove  excess  of  stain 
id  to  differentiate,  10  minutes  ;  then  dehydrate  by  the  ascend- 
ing series  of  alcohols  included  by  h  toy  as  above."  (Mic.  Bui. 
and  Science  News,  April,  1894.) 

Miller's    scheme   for    imbedding,    sectioning,    staining,   and 
mounting :  — 


I.   When  Paraffin  is  used 

80  per  cent  alcohol  (a) . 
Stain  (£). 
fash. 

lute  alcohol. 

&. 
Paraffin. 
Imbed. 
Section. 

Fix  on  slide  (</). 
Xylol  00. 
Absolute  alcohol. 
Stain  (£). 
Wash. 

Absolute  alcohol. 
Xylol. 
Balsam. 


2.    When  Celloidin  is  used : 

80  per  cent  alcohol. 

Absolute  alcohol. 

Alcohol  and  ether. 

Dilute  celloidin. 

Saturated  celloidin. 

Imbed. 

80  per  cent  alcohol  (/). 

Section. 

Stain  O). 

Wash.     (Then  glycerin  will  terminate 

if  used  here.) 
95  per  cent  alcohol. 
Eosin-alcohol  (#•). 
Oil    of  origanum  cretici ;    or   oil  of 

cloves  (/&). 
Balsam. 


"  (a)  If  sections  are  to  be  stained  on  the  slide  after  imbed- 
ding in  paraffin,  pass  at  once  to  absolute  alcohol ;  if  the  speci- 
men is  to  be  stained  in  toto,  pass  to  stain,  etc. 

"  (f)  For  staining  in  toto  use  Grenacheri's  borax-carmine, 
alum-carmine,  or  Delafield's  hgematoxylin.  For  staining  on  the 
slide,  use  either  the  above,  or,  preferably,  some  one  of  the  anilin 
colors.  Specimens  hardened  in  chromic  or  osmic  acid  mixtures 
ike  carmine  stains  badly. 


70         BIOLOGICAL  LABORATORY  METHODS 

"  (V)  Clear  in  either  cedar  oil,  beech  wood,  creosote,  or  chloro- 
form. Avoid  clove  oil,  as  it  makes  the  paraffin  granular. 

"  (d)  If  the  specimen  has  been  stained  in  toto,  use  Shalli- 
baum's  collodion  clove-oil  fixative ;  if  sections  are  to  be  stained 
on  the  slide,  use  Mayer's  albumen  fixative. 

"  (e)  If  the  specimen  was  stained  in  toto,  pass  at  once  to  bal- 
sam ;  but  if  sections  are  to  be  stained,  pass  to  absolute  alcohol, 
stain,  etc. 

"  (/)  After  imbedding  and  previous  to  placing  in  80  per  cent 
alcohol,  it  is  well  to  place  the  block  for  a  short  time,  1-2  hours, 
in  chloroform.  This  prevents  the  formation  of  bubbles  and 
makes  the  celloidin  more  uniform  in  consistency. 

"  (g)  For  routine  work,  use  Delafield's  haematoxylin  and  eosin- 
alcohol ;  this  gives  a  double  stain.  Any  other  stain  may  be  used, 
but  some  anilins  color  the  celloidin  intensely.  If  other  stain 
than  the  haematoxylin  is  used,  the  eosin-alcohol  may  be  omitted. 

"  (ti)  If  it  is  desired  to  remove  the  celloidin  from  section, 
clean  the  specimen  in  clove  oil.  With  delicate  section  it  should 
not  be  used."  (Amer.  Man.  Mic.  Jour.,  June,  1894.) 

Staining  in  bulk  (method  after  P.  A.  Fish    in  Proceedings  of  American 

Microscopical  Society}  :  — 

1.  Fixation  of  tissue. 

2.  Staining. 

3.  Dehydrating. 

PARAFFIN  METHOD  CELLOIDIN  METHOD 

4.  Cedar  oil  or  chloroform.  Ether-alcohol. 

5.  Paraffin  imbedding.  Celloidin  imbedding. 

6.  Cutting  sections.  Clearer  (castor-thyme  oil). 

7.  Fixing  to  slide.  Cutting  sections. 

8.  Xylol.  Mounting  in  Canada  balsam. 

9.  Mounting  in  Canada  balsam. 

Section  staining :  — 

1.  Fixation  of  tissue. 

2.  Dehydrating. 

PARAFFIN  METHOD  CELLOIDIN  METHOD 

3.  Cedar  oil  or  chloroform.  Ether-alcohol. 

4.  Paraffin  imbedding.  Celloidin  imbedding. 


PREPARATION  OF  THE  TISSUE  FOR  MOUNTING          /I 

PARAFFIN  METHOD  CELLOIDIN  METHOD 

5.  Cutting  sections.  Castor-thyme  oil. 

6.  Fixing  to  slip.  Cutting  sections. 

7.  Xylol.  Fixing  to  slip  (ether-alcohol). 

8.  Treatment  with  alcohol.  Treatment  with  alcohol. 

9.  Treatment  with  water.  Treatment  with  water. 

10.  Stain. 

11.  Water. 

12.  Wash  in  alcohol. 

13.  Clearer. 

14.  Mounting  in  Canada  balsam. 

All  methods  have  the  same  general  purpose  and  differ  only 
in  minor  details,  and  these  details  are  controlled  entirely  by  the 
purpose  in  view  and  the  character  of  the  object  to  be  cut. 

Taking  the  scheme  mentioned  on  previous  pages  as  our  guide, 
we  will  now  proceed  to  give  in  detail  the  processes  required  to 
prepare  and  mount  the  section  of  the  specimen  for  permanent 
preservation. 

Killing,  fixing,  and  hardening.  —  The  three  steps,  killing, 
fixing,  and  hardening,  may  be  accomplished  in  some  cases  by  the 
use  of  one  fluid,  but  in  others  it  will  require  the  use  of  two  or 
three  distinct  solutions  to  reach  the  desired  results.  The  killing 
should  be  done  as  quickly  as  possible  to  prevent  the  collapsing 
of  the  tissues  and  otherwise  injury  to  the  cells  and  their  contents. 
The  prime  object  of  the  fixing  agent  is  to  set  the  tissues  in  their 
natural  forms  as  they  appear  during  life ;  and  the  hardening 
agent  is  to  give  strength  and  tenacity  to  the  cells  for  subsequent 
treatment. 

Usually  the  same  agent  is  adapted  to  both  killing  and  fixing. 
It  is  best  to  have  the  material  in  small  pieces,  so  that  the  fixing 
agent  will  penetrate  thoroughly  into  all  portions  of  the  specimen. 
The  volume  of  the  agent  should  be  about  15  to  25  times  greater 
than  the  specimen  to  be  treated.  All  fixing  agents  except  alcohol 
must  be  washed  out  of  the  specimen  before  the  succeeding 
steps  are  taken,  because  precipitates  are  often  produced  which 
greatly  interfere  with  the  proper  examination  of  the  tissues  if 
this  is  not  done.  All  aqueous  agencies  are  washed  out  with 


72         BIOLOGICAL  LABORATORY  METHODS 

running  water,  while  all  alcohol  fixing  agents  are  washed  with 
alcohol  of  the  same  strength  as  the  agent.  This  washing  out  takes 
from  twelve  to  twenty-four  hours,  but  the  time  may  be  shortened 
by  keeping  the  fluid  lukewarm. 

The  following  are  some  of  the  most  important  killing  and 
fixing  agents :  — 

KILLING  FLUID  FOR  PLANT  TISSUE  (Schaffner's  formula)  :  — 

Chromic  acid 0.8  cc. 

Glacial  acitic  acid          ....         0.5  cc. 
Water 99.0  cc. 

Sixty  cc.  of  this  solution  will  be  sufficient  to  kill  one  or  two 
dozen  objects  of  small  size.  The  specimens  should  be  immersed 
in  the  agent  as  soon  as  they  are  cut,  and  must  be  kept  in  it  from 
twelve  to  twenty-four  hours.  This  fluid  is  washed  out  by  soaking 
in  water  from  one  to  two  hours  in  several  changes. 

Chromic  acid  and  its  combination  with  other  chemicals  give 
the  most  valuable  killing  and  fixing  agents  for  botanists.  It  is 
difficult  to  give  exact  formulae  for  this  agent,  because  opinions 
differ  as  to  the  effects  produced  on  the  plants,  and  it  is  best  to 
try  the  experiment  to  determine  the  strength  of  the  fluid  needed. 
But  the  general  formula  given  above  may  be  relied  upon  for 
favorable  results  in  most  cases.  This  agent  tends  to  harden  the 
specimen,  and  some  difficulty  may  be  experienced  in  cutting  the 
sections.  Under  such  conditions  it  will  be  best  to  use  another 
killing  and  fixing  solution. 

In  the  use  of  chromic  acid  it  must  be  borne  in  mind  that  the 
agent  combines  chemically  with  the  tissues,  and  the  proper  stain, 
therefore,  to  apply  to  the  sections  is  haematoxylin  or  an  anilin 
dye.  Osmic  acid  produces  the  same  results  and  must  be  followed 
with  the  anilin  stains. 

PERENYI'S  FORMULA:  — 

Nitric  acid 40  cc. 

Chromic  acid 30  cc. 

Alcohol 3°  cc- 

Any  alcoholic  stain  may  follow  this  fixing  agent. 


PREPARATION  OF  THE  TISSUE   FOR  MOUNTING          73 

FLEMMING'S  CHROMIC  ACID  SOLUTION:  — 

Chromic  acid  of  I  per  cent 25  vols. 

Osmic  acid  of  I  per  cent 10  vols. 

Acetic  acid  of  I  per  cent         .         .         .         .         .  10  vols. 

Water 55  vols. 

This  formula  is  the  stock  solution.  Lee  in  speaking  of  this 
fixing  agent  says,  "  I  recommend  that  Flemming's  mixture 
should  be  used  wherever  it  is  possible,  as  I  believe  it  to  be  in 
general  far  the  best  fixing  agent  yet  invented,  with  the  exception 
of  Hermann's  mixture,  which  is  unfortunately  too  expensive  to 
be  employed  in  large  quantities."  The  above  formula  is  the 
weak  solution,  but  Flemming  gives  the  following,  which  he  terms 
his  strong  solution. 

Chromic  acid  I  per  cent 15  parts 

Osmic  acid .         .  4  parts 

Glacial  acetic  acid I  part 

Lee  states  that  on  account  of  the  large  amount  of  organic  acid 
given  in  these  two  formulae  they  may  not  keep  well,  and  he 
recommends  that  in  the  first  formula  the  osmium  be  kept  in 
2  per  cent  solution  in  chromic  acid  of  i  per  cent,  and  that  20 
volumes  of  chromic  acid,  5  volumes  of  osmium  solution,  and  65 
volumes  of  water  be  taken  to  form  the  solution  when  required. 
In  the  last  formula  he  recommends  that  it  be  made  up  from 
time  to  time  from  stock  solutions,  in  which  the  osmium  is  kept 
separate  from  the  acetic  acid.  In  this  case  it  will  require  two 
separate  solutions  as  follows  :  — 

%     A.   Chromic  acid  I  per  cent II  parts 

Glacial  acetic  acid I  part 

Distilled  water 4  parts 

B.  Two  per  cent  solution  of  osmium  in  I  per  cent  chromic  acid  solution; 
and  when  required,  mix  four  parts  of  A  with  one  of  B. 

HERMANN'S  FORMULA,  MODIFIED  BY  LEE:  — 

Platinic  chloride  I  per  cent  .         .         .         .          15  parts 

Glacial  acid  I  per  cent  .....  I  part 

Osmic  acid  2  per  cent  .         .         .         .         .  4  or  2  parts 


74  BIOLOGICAL  LABORATORY  METHODS 

Protoplasmic  structures  are  claimed  by  Hermann  to  be  well 
preserved  in  this  solution,  better  than  in  chromic  mixtures.  It 
does  not  cause  precipitates  as  was  stated  with  the  chromic 
mixtures.  Lee  says,  "  On  the  whole,  I  take  it  that  this  mixture 
\^  probably  the  most  fixative  yet  discovered."  The  objection  to  it 
is  the  great  expense  in  its  preparation. 

MERKEL'S  FORMULA  (a  very  delicate  fixative)  :  — 

Chromic  acid  and  platinic  chloride,  equal  volumes  of  1 : 400  solution  in 
water. 

This  agent  was  originally  introduced  for  the  study  of  karyo- 
kinesis  (the  transformations  of  the  nucleus  during  cell-division), 
but  it  is  excellent  for  general  use  in  the  case  of  delicate  objects. 
It  is  readily  washed  out  with  50  to  70  per  cent  alcohol.  Stain 
with  safranin  or  Kleinenberg's  haematoxylin. 

KLEINENBERG'S  FORMULA  :  — 

Picric  acid  (saturated  solution  in  water)       .         .         100  parts 
Sulphuric  acid,  concentrated        .         .         .  2  parts 

Filter. 

Add  three  times  bulk  of  water. 

Add  as  much  creosote  as  will  mix. 

This  agent  will  kill  quickly,  remove  sea-water  entirely,  and 
may  be  wholly  replaced  by  alcohol.  It  has  been  much  used  at 
the  Naples  Aquarium.  It  is  claimed  by  some  to  be  superior  to 
chromic  acid  solutions,  because  it  does  not  produce  precipitates, 
but  leaves  the  tissues  in  good  condition  for  staining.  Large 
quantities  of  the  fluid  should  be  used,  and  it  must  be  changed  as 
often  as  it  becomes  turbid. 

PICRIC  ALCOHOL  :  — 

Alcohol 250  parts 

Picric  acid .  I  part 

Water 250  parts 

Fix  for  twenty-four  hours,  and  then  wash  out  with  alcohol, 
soaking  for  a  day  in  70  per  cent  and  then  a  day  in  80  per  cent 
alcohol.  It  is  best  to  use  saturated  solutions  of  the  picric 


PREPARATION  OF  THE  TISSUE  FOR  MOUNTING          75 

acid,  because  the  dilute  solutions  are  more  apt  to  macerate  the 
specimen.  This  fixing  agent  has"  great  penetrating  power,  and 
yet  it  can  be  soaked  out  with  alcohol,  and  tissues  fixed  in  it 
may  be  perfectly  stained  in  any  dye. 

PICRO-SULPHURIC  ACID  (Kleinenberg's  and  Mayer's  formula)  :  — 

Distilled  water 100  vols. 

Sulphuric  acid -  .       :.  2  vols. 

Picric  acid,  as  much  as  will  dissolve        .          (about  0.25  vols.). 

The  above  is  the  concentrated  solution.  Kleinenberg's 
formula  is  obtained  by  diluting  with  three  times  its  volume  of 
water,  and  this  is  the  form  generally  employed.  Permit  the 
specimen  to  remain  in  the  fixing  solution  for  three  or  more 
hours,  and  then  place  it  in  70  per  cent  alcohol  for  five  or  six 
hours  to  remove  the  acid  and  harden  the  tissues  ;  remove  to  90 
per  cent  alcohol  and  change  again  until  the  yellow  stain  due  to 
the  acid  is  entirely  eliminated.  Always  wash  out  with  alcohol. 
The  specimens  come  out  of  this  fixing  agent  with  but  little  if 
any  shrinkage. 

RIPART'S  AND  PETIT'S  CHLORIDE  AND  ACETATE  OF  COPPER  FIXING 
SOLUTION  :  — 

Copper  chloride     .  .  .  .  .  ,     ,        0.39  grammes 

Copper  acetate       .  .  .  ,  .         . '       0.30  grammes 

Acetic  acid  crystals  .  ,  .  ...         I       gramme 

Camphor  water      .  .  .  .  .        -'75       grammes 

Distilled  water        .  .  .  .         •       75       grammes 

Lee  considers  this  solution  a  most  valuable  agent  for  fixing 
cytological  specimens,  and  it  has  the  excellent  quality  of  yield- 
ing fine  results  when  followed  with  the  stain  methyl-green ;  the 
staining  is  almost  instantaneous.  The  solution  is  specially 
recommended  for  those  experiments  where  the  tissues  are  fresh 
and  hardening  is  not  a  required  property.  The  osmium  is 
added  to  the  solution  to  strengthen  the  fixing  action. 

Formalin  is  one  of  the  recent  fixing  agents,  and  is  an  excel- 
lent preservative.  Wash  out  with  water  and  follow  by  any 
stain. 


76  BIOLOGICAL  LABORATORY   METHODS 

Bichlorid  of  mercury.  —  This  is  a  fixing  agent  of  a  high  order, 
and,  when  properly  applied,  the  tissues  will  yield  to  stains  of 
any  nature.  In  the  case  of  carmine  a  brilliant  color  is  produced 
because  of  the  action  of  the  mercury  on  the  stain  resulting  in 
the  formation  of  mercuric  carminate.  There  are  a  number  of 
combinations  with  corrosive  sublimate,  but  Lee  strongly  en- 
dorses a  saturated  solution  in  five  per  cent  acetic  acid,  which  he 
has  found  to  be  an  excellent  formula  for  marine  animals.  The 
most  concentrated  solution  possible  must  be  taken  in  every 
instance. 

Do  not  place  iron  or  steel  instruments  in  contact  with  this 
agent,  because  precipitates  will  be  formed  which  are  injurious  to 
the  tissues.  Wood,  glass,  or  platinum  can  be  used.  The  speci- 
men must  remain  in  this  solution  only  long  enough  to  fix,  which 
may  be  determined  by  the  degree  of  opaqueness  the  tissue 
assumes,  thus  showing  the  penetrating  power  of  the  corrosive 
sublimate.  The  time  of  fixing  depends  on  the  size  of  the  object, 
extending  from  a  few  minutes  to  a  much  longer  period.  Wash 
out  with  either  water  or  alcohol.  Be  careful  to  remove  all  trace 
of  the  sublimate,  or  otherwise  the  tissues  will  become  brittle. 

Alcohol.  —  There  are  two  grades  of  this  agent,  absolute  and 
alcohol  diluted  with  two-thirds  water.  Use  this  fixing  agent 
with  tissues  which  are  not  apt  to  shrink  easily.  Absolute  alco- 
hol has  a  great  affinity  for  water,  and  in  the  rapid  dehydrating 
the  delicate  cells  will  collapse  if  delicate  organisms  are  placed 
in  it.  A  very  large  proportion  of  the  alcohol  must  be  employed 
for  fixation. 

Specimens  to  be  examined  for  fatty  matter  should  not  be 
treated  with  alcohol,  because  this  reagent  dissolves  from  the 
tissues  a  portion  of  the  fat,  and  therefore  in  the  case  of  fatty 
degeneration  it  is  best  to  examine  the  specimen  in  the  fresh 
state.  It  is  recommended  by  pathologists  that  it  is  best  to  use 
alcohol  only  in  those  cases  where  rapid  diagnosis  is  demanded. 

Hardening.  —  The  methods  of  cutting  in  celloidin  and  in  par- 
affin have  now  become  so  general  among  microscopists,  soft 
tissues  can  be  sectioned  in  the  microtome,  in  most  instances, 


PREPARATION  OF  THE  TISSUE  FOR  MOUNTING          77 

without  first  treating  them  in  the  hardening  fluids,  which  was 
always  resorted  to  before  the  imbedding  methods  became  so 
universal.  In  a  few  instances  hardening  is  required,  and  it  is 
important,  therefore,  that  the  well-informed  microscopist  should 
know  how  to  harden  as  well  as  how  to  imbed  in  paraffin  or  in 
celloidin.  , 

Hardening  should  not  be  hastened,  but  the  action  should  be 
slow,  so  as  to  prevent  shrinking,  and  do  not  overharden.  A 
large  proportion  of  the  agent  must  be  employed,  and  the  object 
must  be  suspended  in  it  by  means  of  a  cord,  so  that  any  precipi- 
tates which  may  form  will  fall  to  the  bottom  of  the  containing 
vessel  and  adhere  to  the  specimen.  Frequent  changing  of  the 
fluid  will  be  beneficial.  If  a  small  portion  of  the  hardening 
agent  be  employed,  it  will  soon  become  saturated  with  the 
soluble  matters  in  the  object,  and  the  hardening  action  will  stop, 
followed  by  maceration  of  the  tissues ;  hence  the  importance  of 
using  large  portions  of  the  fluid  and  in  frequent  changes. 

Some  of  the  best  hardening  agents  are  as  follows  :  — 

EHRLICH'S  FLUID  (after  Miiller)  :  — 

Bichromate  of  potash 2j  parts 

Sulphate  of  copper          .         .         .         .•                 .         I    part 
Water  100    parts 

This  solution  will  yield  to  the  action  of  alcohol,  and  after  the 
specimen  has  hardened  it  must  be  washed  from  four  to  ten  days 
in  alcohol.  This  solution  is  very  good  for  soft  tissue  and  where 
much  blood  is  present.  After  transferring  to  the  alcohol,  place 
the  vessel  in  the  dark,  because  light  develops  crystals  on  the  sur- 
face of  the  alcohol,  and  its  penetrating  properties  are  greatly 
reduced. 

MERKEL'S  FORMULA:  — 

Equal  volumes  of  i  :  400  solution  of  chromic  acid  and  platinic  chloride. 

This  formula  is  excellent  for  delicate  objects,  and  it  acts 
slowly,  so  that  the  specimens  should  remain  in  it  for  several 
days.  Merkel  allowed  about  four  days  for  it  to  harden  the 


78  BIOLOGICAL   LABORATORY  METHODS 

retina.  Wash  out  with  alcohol  of  about  70  per  cent.  Staining 
acts  well  after  this  hardening  agent. 

Alcohol,  —  Begin  with  a  weak  alcohol  and  follow  with  stronger 
until  absolute  strength  is  reached.  This  method  will  prevent 
too  rapid  action  of  the  hardening  agent,  and  shrinkage  of  the 
tissue  will  not  result.  Alcohol  is  not  as  good  a  hardening  agent 
as  either  one  of  the  above,  but  it  will  give  valuable  results  when 
it  follows  the  above  formulae.  Pollen,  starch,  yeast  cells,  and 
spores  after  hardening  in  alcohol  may  be  mounted  in  glycerin 
jelly  with  satisfactory  results.  After  hardening  place  them  in 
dilute  glycerin  and  then  mount  in  the  jelly. 

One  of  the  disadvantages  in  the  use  of  alcohol  as  a  hardening 
agent  is  that  it  coagulates  the  albuminoids  which  produce  a 
reduction  in  the  transparency  of  the  specimen. 

In  some  specimens,  like  muscles  and  lung  tissues,  the  alcohol 
does  not  harden,  even  after  being  immersed  for  a  long  period  of 
time ;  but  in  these  cases,  if  the  specimens  are  placed  first  in 
dilute  gum  Arabic  for  twenty-four  hours  and  then  in  the  alcohol, 
the  acacia  is  precipitated,  and  then  the  tissues  harden.  The 
gum  can  be  removed  by  water. 

Van  Gieson *  recommends  the  use  of  formalin  as  a  hardening 
agent  for  brain  and  spinal  cord.  The  solutions  are  made  from 
4  to  10  per  cents,  and  the  specimens  are  soaked  ten  days.  Fol- 
low with  alcohol  in  successive  strengths. 

Bacteria  are  best  hardened  in  alcohol,  because  such  agents, 
like  Miiller's  fluid,  produce  dark  granulations  which  are  difficult 
to  eliminate.  Clear  up  by  means  of  strong  acetic  acid. 

Dehydrating.  —  The  water  found  in  all  tissue  is  very  deleteri- 
ous, not  only  to  the  free  penetration  of  the  imbedding  material 
required  in  cutting  certain  tissues,  but -its  presence  will  cause 
the  tissue  to  decay.  Water  also  prevents  the  free  penetration 
of  balsam,  which  is  the  preserving  medium  and  in  which  the 
specimen  must  be  finally  mounted.  The  usual  method  of  pro- 
cedure is  as  follows  :  — 

The  specimen  is  placed  in  rather  diluted  alcohol  for  a  short  time 

1  Anat.  Anzcig.,  X,  1895. 


! 


PREPARATION  OF  THE  TISSUE  FOR  MOUNTING          79 

and  then  transferred  to  a  vessel  containing  a  stronger  quantity 
of  the  spirit,  and  so  on  until  95  per  cent  alcohol  has  been  used. 
By  pursuing  this  course  of  treatment  there  is  slight  danger  of 
causing  the  specimen  to  shrink.  Some  manipulators  use  abso- 
lute alcohol  for  the  last  washing,  but  this  is  not  deemed  necessary 
in  most  cases,  since  the  small  amount  of  water  remaining  in  the 
specimen  after  floating  for  a  while  in  95  per  cent  alcohol  will  be 
removed  by  the  clearing  agent. 

In  those  cases  where  a  fixing  agent  has  been  used,  it  is  first 
important  to  remove  this  fixing  agent  before  dehydrating  is  per- 
formed, unless  alcohol  is  the  removing  agent,  and  then  in  that 
case  it  will  partially  dehydrate  while  it  is  removing  the  super- 
fluous fixing  substance.  Care  must  be  exercised  in  performing 
this  part  of  the  operation  not  to  prolong  the  dehydration  too  far, 
because  the  alcohol  will  cause  the  tissues  to  become  hard, 
brittle,  and  eventually  shrivel  up  into  an  unnatural  form. 

Some  authorities  recommend  the  use  of  95  per  cent  alcohol 
at  once ;  and  it  is  claimed  that  the  weaker  solutions  of  alcohol 
are  not  necessary,  because  if  a  large  quantity  of  the  strong 
alcohol  is  used,  the  water  is  washed  out  by  the  flooding  of  the 
dehydrating  agent  before  the  shrinking  tendency  is  transmitted 
to  the  tissue.  By  using  large  quantities  of  95  per  cent  alcohol 
the  diffusion  currents,  which  do  so  much  damage  to  the  tissues, 
are  greatly  lessened  by  the  quick  action  of  the  surplus  quantity 
of  the  dehydrating  agent.  Most  microscopists,  however,  prefer 
to  use  the  alcohol  in  different  strengths  as  stated  above,  so  as  to 
avoid  all  chance  of  the  violent  osmosis  which  would  cause  a  ruin- 
ous contraction  of  the  delicate  tissue.  The  specimen  should  re- 
main in  each  degree  of  the  alcohol  from  four  to  twenty-four  hours. 

Glycerin  has  been  used  by  Overton  for  dehydrating,  and  he 
claims  that  the  cells  will  not  contract.  This  method  consists 
in  placing  the  specimen  in  10  per  cent  glycerin,  which  solution 
is  exposed  to  the  action  of  the  atmosphere  when  the  gradual 
vaporation  of  the  water  will  so  concentrate  the  glycerin  as  to 
permit  of  the  transfer  of  the  specimen  from  it  to  strong  alcohol 
without  the  danger  of  shrinkage. 


80        BIOLOGICAL  LABORATORY  METHODS 

Clarifying. —  After  the  tissues  have  been  thoroughly  dehydrated, 
it  is  then  necessary  to  remove  the  alcohol  by  replacing  it  with 
some  agent  which  is  readily  miscible  with  the  reagent  used  in 
the  imbedding  process,  and  this  agent  must  also  be  anhydrous. 
This  process  is  called  clearing  or  clarifying.  If  the  alcohol 
remains  in  the  specimen,  it  will  seriously  interfere  with  the  per- 
meation of  the  imbedding  substance  and  will  seriously  prevent 
the  penetration  of  the  balsam  which  is  used  to  permanently  pre- 
serve the  tissue.  Another  object  sought  in  clarifying  is  to 
render  the  specimen  transparent. 

It  is  very  important,  especially  in  the  case  of  delicate  objects, 
that  the  transition  from  the  dehydrating  agent  to  the  clearing 
agent  should  be  gradual,  so  that  the  tissues  will  not  shrink  but 
remain  in  their  normal  condition.  Dr.  Gierbrechts  recpmmends 
the  following  method  he  has  found  to  be  efficient  for  accom- 
plishing this  gradual  substitution  of  the  clearing  agent  for  the 
alcohol.  A  staining  glass  is  partly  filled  with  the  clarifying 
fluid,  and  alcohol  is  placed  on  top  of  it.  The  difference  in  the 
specific  gravity  will  cause  the  alcohol  to  remain  on  top  for  some 
time.  The  tissue  is  now  placed  in  the  vessel,  and  it  will  slowly 
sink  through  the  alcohol  into  the  clearing  agent,  and  the  former 
will  slowly  give  place  to  the  latter.  The  alcohol  is  now  drawn 
off  by  means  of  a  pipette.  The  specimen  must  remain  in  the 
clarifying  agent  until  thoroughly  clear ;  this  will  require  from  a 
few  minutes  to  an  hour,  and,  in  a  few  instances,  to  several  hours, 
the  length  of  time  being  governed  by  the  character  of  the 
object. 

The  following  are  some  of  the  clearing  agents  used  by  the 
best  microscopists :  — 

Anilin  oil.  Clove  oil. 

Bergatnot  oil.  Origanum  oil. 

Cedar  oil.  Turpentine  oil. 

Anilin  oil  is  a  strong  agent,  and  it  also  acts  quite  well  as  a 
dehydrating  agent.  It  will  extract  water  from  specimens  which 
have  been  treated  with  70  per  cent  alcohol.  For  certain  kinds 


PREPARATION  OF  THE  TISSUE  FOR  MOUNTING 


81 


of  work  it  will  be  found  exceedingly  valuable  —  particularly  for 
preparing  specimens  for  paraffin  imbedding.  Unless  this  agent 
is  removed  by  soaking  for  several  hours  in  chloroform,  however, 
the  sections  will  turn  yellow  after  a  while. 

Oil  of  bergamot  does  not  destroy  the  anilin  colors  placed  in 
the  tissues,  and  it  has  the  additional  valuable  property  of  clear- 
ing the  specimens  very  quickly  which  have  been  dehydrated  in 
95  per  cent  alcohol. 

Oil  of  cedar  must  be  thin  in  consistency  and  of  a  light  yellow 
color  to  give  the  best  results.  This  oil  evaporates  slowly  and 
mixes  well  with  balsam  dissolved  in  chloroform.  It  clears  speci- 
mens very  wrell  and  readily  which  have  been  dehydrated  in  95 
per  cent  alcohol.  Mr.  Lee  says,  in  reference  to  this  oil,  "A 
laboratory  equipped  with  cedar  oil  and  origanum  oil  is  fully 
equipped  for  all  possible  cases  (the  origanum  oil  being  used 
merely  to  take  the  place  of  cedar  wood  oil  for  the  special  case  of 
celloidin  sections)."  Cedar  oil  clears  tissues  stained  in  anilin 
dyes  without  extracting  the  color. 

Oil  of  cloves  is  a  very  good  clearing  agent  for  certain  kinds  of 
work  where  minute  dissection  is  resorted  to.  The  specimen, 
however,  cleared  in  it  is  very  apt  to  become  brittle  after  a  short 
soaking  in  the  oil,  and  this  feature,  in  many  cases,  will  be  a 
decided  disadvantage.  The  tendency  to  form  minute  drops 
over  the  slide  gives  it  a  value  for  minute  dissection.  Mixing  it 
with  oil  of  bergamot  will  reduce  its  tendency  to  produce  brittle- 
ness  in  the  tissues.  It  is  an  excellent  clearer,  and,  when  prop- 
erly used,  will  be  found  to  be  a  valuable  addition  to  the 
laboratory  equipment. 

Oil  of  origanum.  —  This  oil  is  valuable,  since  it  will  clear  speci- 
mens which  have  been  stained  in  anilin  dyes  without  affecting 
the  colors  to  any  serious  degree.  It  does  not  volatilize  easily 
and  rapidly,  and  is  unchanged  when  exposed  to  the  action  of  the 
light.  The  odor  is  not  disagreeable.  Celloidin  sections  are 
quickly  cleared  by  it  without  dissolving  the  celloidin. 

Oil  of  turpentine  is  only  valuable  for  clearing  specimens  after 
they  have  been  cut  in  paraffin.  Its  disadvantage  consists  in  the 


82         BIOLOGICAL  LABORATORY  METHODS 

shrinkage  it  gives  the  cells,  and  in  some  instances,  where  alco- 
hol is  present,  altering  even  the  structure. 

Infiltrating.  —  Paraffin  with  a  melting  point  of  35°  is  melted 
over  the  water-bath  and  kept  for  some  time  heated  only  at  its 
melting  point.  The  specimen  to  be  imbedded  is  placed  in  a 
solution  of  paraffin  mixed  with  the  clearing  agent,  and  is  then 
transferred  into  the  hot  paraffin,  and  here  it  is  left  until  the 
paraffin  penetrates  all  portions  of  the  tissue.  This  is  termed 
infiltrating.  Generally  it  takes  about  one  hour  to  properly 
infiltrate. 

Of  course  if  celloidin  is  to  be  used  in  the  place  of  paraffin 
the  infiltrating  must  be  performed  with  the  celloidin  as  the 
agent.  The  alcohol  is  removed  in  this  case  by  ether-alcohol, 
equal  volumes  of  sulphuric  ether  and  95  per  cent  alcohol,  as 
the  clarifying  agent,  and  from  the  vessel  containing  the  ether- 
alcohol  the  specimen  is  transferred  to  a  thin  celloidin  solution  (\\ 
grammes  of  collodion  cotton  dissolved  in  100  cc.  of  ether-alcohol), 
where  it  must  remain  for  two  or  three  hours,  depending  upon 
the  character  of  the  specimen.  It  is  then  transferred  to  thick 
celloidin  (6  grammes  of  collodion  cotton  dissolved  in  i  oo  cc. 
ether-alcohol).  In  this  solution  the  specimen  is  left  for  three 
to  five  hours. 


CHAPTER   V 


IMBEDDING   METHODS 

THE  clearing  process  described  in  the  preceding  chapter  is 
preliminary  to  the  imbedding  operation.  This  clearing  work  is 
necessary  before  the  object  is  imbedded  in  paraffin,  because 
otherwise  the  paraffin  would  refuse  to  enter  thoroughly  into  all 
portions  of  the  mass,  so  that  proper  cutting  with  the  microtome 
would  become  difficult  if  not  impossible.  It  is,  therefore, 
important  that  the  clearing  work  be  correctly  and  thoroughly 
done,  in  order  to  secure  satisfactory  results  with  the  imbedding 
material.  The  oil  used  in  the  process  must  penetrate  all  por- 
tions of  the  mass,  and  the  water  must  be  entirely  eliminated, 
and  the  clearing  medium  must  also  mix  well  with  the  paraffin 
used  in  the  imbedding  work.  With  these  simple  facts  well  borne 
in  mind,  the  student  will  proceed  as  follows  to  complete  the 
imbedding  of  the  specimen  preparatory  to  cutting  the  sections. 

Imbedding  in  paraffin.  —  This  is  the  method  for  cutting  small 
objects.  Melted  paraffin  is  substituted  for  the  clearing  sub- 
stance by  immersing  the  specimen  in  a  vessel  containing  paraffin 
of  the  lowest  melting  point.  The  specimen  is  kept  in  the  im- 
bedding mass  until  it  becomes  thoroughly  impregnated  with  it 
(infiltrating).  The  paraffin  should  be  placed  in  one  of  the 
standard  water-baths  and  kept  just  at  the  melting  point.  Dura- 
tion of  bath  will  depend  on  the  nature  of  the  specimen  and  the 
character  of  the  clearing  agent  employed.  Prolonged  heating  at 
any  time  is  injurious  to  many  forms  of  vegetation,  and  it  should 
therefore  be  borne  in  mind  that  the  employment  of  paraffin 
of  low  melting  point  and  the  reduction  in  the  degree  of  heat, 
shortening  as  much  as  possible  the  time  of  the  bath,  will  secure 
the  best  results. 

83 

'    UNIVERSITY 

OF 


84  BIOLOGICAL   LABORATORY   METHODS 

After  saturation  has  been  accomplished,  the  specimen  is  trans- 
ferred to  the  melted  paraffin  contained  in  the  imbedding  box  de- 
scribed on  page  64  and  illustrated  in  Figs.  55  and  56,  or  in  some 
similar  box  for  the  purpose,  arranged  as  desired,  and  then  cooled 
with  water  as  soon  as  possible.  Care  should  be  exercised  not 
to  permit  the  water  to  enter  into  the  warm  paraffin,  because  if  it 
does,  the  mass  will  at  once  become  filled  with  holes  and  thus  be 
injured  for  yielding  good  results  in  the  microtome.  As  soon  as 
cooled,  the  mass  is  shaped  into  a  rectangular  form,  so  that  when 
placed  in  the  microtome  one  face  of  the  mass  will  be  square  with 
the  knife  and  the  opposite  edge  parallel  with  it.  The  microtome 
knife  must  not  be  moistened  with  liquid  of  any  kind,  but  kept 
dry  during  the  entire  time  of  cutting  the  sections,  so  that  rolling 
of  the  paraffin  will  be  largely  prevented.  The  temperature  of  the 
room  should  be  made  to  sustain  a  certain  relationship  to  that  of 
the  boiling  point  of  the  paraffin.  In  winter,  when  the  melting 
point  of  the  paraffin  is  45°  C.,  the  temperature  of  the  laboratory 
should  be  between  15°  and  17°  C.;  in  summer  the  relationship 
should  be,  for  paraffin  48°,  and  22°  for  the  laboratory.  The  tem- 
perature of  the  paraffin,  in  winter,  may  be  controlled  by  means  of 
a  reflector  lamp,  so  placed  as  to  have  the  heat  rays  reflected  on 
the  sections  as  they  are  being  cut ;  in  the  summer  this  lamp  may 
be  replaced  by  a  block  of  ice,  with  the  reflector  projecting  the 
cool  rays  upon  the  mass  of  paraffin ;  or  the  room  may  be  regu- 
lated in  temperature  by  the  adjustment  of  the  window  fixtures. 

If  it  is  desirable  to  produce  in  the  cutting  what  is  termed 
"  ribbon-sections,"  or  in  series,  it  will  be  best  to  have  in  the 
laboratory  one  of  the  microtomes  made  for  that  purpose,  such 
as  Minot's  automatic  microtome,  or  any  other  of  the  same  char- 
acter; but  it  is  possible  to  make  these  ribbons  with  the  judi- 
cious use  of  the  knife  on  any  of  the  ordinary  standard  microtomes. 
The  following  hints  are  given  to  aid  the  student  in  accomplish- 
ing this  end :  — 

i .  The  edge  of  the  section  is  held  down  by  means  of  a  camel's- 
hair  pencil  until  contact  is  made  with  the  next  section  and  the 
heat  from  the  lamp  causes  it  to  cohere. 


IMBEDDING  METHODS  85 

2.  The  block  of  paraffin  must  be  pared  down  very  close  to 
the  object  and  so  cut  as  to  present  a  square  edge  to  the  knife. 
The  cutting  must  be  rapid,  so  that  the  blows  of  the  knife  will 
slightly  heat  the  paraffin,  and  thus  cause  the  edges  of  the  sec- 
tions to  cohere  and  form  the  ribbon. 

3.  The  use  of  what  is  known  as  the  "  section  stretcher,"  which 
is  so  placed  as  to  rest  on  the  upper  surface  of  the  knife,  and 
holds  the  sections  in  a  flat  position  and  in  contact  with  each  other 
in  the  best  way  to  form  the  chain. 

In  the  case  of  those  specimens  which  are  so  brittle  or  the 
parts  of  which  are  so  slightly  cohering  that  separation  takes 
place  during  the  cutting,  the  following  method  of  procedure  is 
recommended  :  — 

A  bottle  of  collodion  is  placed  convenient  to  the  microtome, 
so  that  a  brush  may  be  dipped  in  it  and  the  surface  of  the 
paraffin  covered  with  a  thin  coating  of  the  collodion  as  each 
section  is  cut.  The  collodion  must  be  of  such  consistency 
as  to  dry  quickly  without  leaving  a  polished  residue  on  the  sec- 
tion. This  coating  must  be  made  very  thin,  or  the  results  will 
not  be  satisfactory.  Repeat  the  operation  after  cutting  each 
section,  and  the  collodion  will  be  found  to  sink  into  the  specimen 
and  upon  drying  will  hold  the  parts  together.  When  the  section 
is  taken  from  the  knife,  it  should  be  transferred  to  the  slide  with 
the  collodion  surface  downward,  in  contact  with  the  slide,  which 
has  been  previously  prepared  with  Scriaillibaum's  fixative.  ("One 
part  of  collodion  is  shaken  up  with  three  to  four  volumes,  accord- 
ing to  the  consistency  of  the  collodion,  of  clove  oil  or  lavender  oil. 
This  should  give  a  clear  solution.  A  little  is  spread  on  a  slide 
with  a  small  brush.  After  arranging  the  sections  on  the  prepared 
surface,  warm  over  a  water-bath,  gently,  until  the  clove  oil  has 
evaporated  —  five  to  ten  minutes."1) 

For  cutting  ribbons  Adam  Sedgwick  proposes  the  use  of  softer 
paraffin  around  the  block  of  harder  paraffin  in  which  the  speci- 
men is  imbedded.  After  the  imbedding  has  been  accomplished, 
the  block  is  dipped  in  softer  melted  paraffin  and  the  surplus 

1  Lee's  "  Microtomist's  Vade-Mecum,"  p.  212. 


86  BIOLOGICAL   LABORATORY   METHODS 

is  cut  off,  leaving  only  a  thin  layer  of  the  coating  on  the  front 
and  rear  faces  of  the  block.  When  the  cutting  begins,  this  soft 
paraffin  will  cause  the  sections  to  unite  into  the  ribbon  form. 

In  the  laboratory  there  should  be  found  two  grades  of  paraffin  : 
one  melting  at  a  temperature  of  45°  to  46°  C.  and  a  hard  par- 
affin melting  at  a  temperature  of  55°  to  56°  C.  The  microscopist 
can  then  mix  these  two  together  in  proportions  to  suit  the  condi- 
tion of  temperature  in  the  room.  It  must  be  remembered  that 
the  harder  the  paraffin  the  thinner  may  the  sections  be  cut,  but 
there  is  stronger  liability  to  roll.  It  is  best,  therefore,  to  mix 
the  two  kinds  in  such  proportions  as  to  produce  a  paraffin  which 
will  give  a  melting  point  of  50°  to  55°  C.  This  will  have  to  be 
determined  by  experiment,  but  ordinarily  the  combination  may 
be  secured  by  mixing  in  the  proportion  of  20:9. 

Plant  tissues  vary  so  much  it  is  necessary  to  use  great  care  in 
their  treatment  to  prevent  shrinkage  while  passing  through  the 
several  liquids  preparatory  to  imbedding  for  paraffin  cutting. 
In  the  case  of  those  specimens  which  contain  much  meristemic 
and  watery  thin-walled  tissue,  the  following  procedure  is  recom- 
mended by  Dr.  J.  W.  Moll  in  the  Botanical  Gazette  for  January, 
1888. 

The  dehydrating  of  the  specimen  was  accomplished  by 
using  a  solution  of  chromic  acid  and  20  per  cent,  35  per  cent, 
50  per  cent,  75  per  cent,  and  90  per  cent  alcohols.  The 
chromic  acid  acted  on  the  protoplasm  so  as  to  fix  it  and  macerate 
the  cellulose,  and  thus  permit  the  alcohol  to  permeate  more  freely. 
The  dehydration  must  be  done  by  slow  degrees,  so  it  is  best  to 
permit  the  specimen  to  remain  in  the  several  alcohols  for  twelve 
to  twenty-four  hours,  depending  upon  the  size  of  the  specimens 
under  treatment.  Passing  from  the  alcohols,  the  plant  is  placed 
in  a  solution  of  equal  parts  of  turpentine  and  paraffin.  This  solu- 
tion is  now  raised  from  a  temperature  of  20°  C.  to  45°  C.  The 
specimens  are  then  placed  in  melted  paraffin,  which  is  retained 
at  a  temperature  of  50°.  It  will  take  from  one  to  two  hours  to 
permeate  the  specimens. 

Mr.  Gustav  Mann,  in  the  "  Transactions  and  Proceedings  of 


IMBEDDING   METHODS  8/ 

the  Botanical  Society  of  Edinburgh,"1  thus  describes  a  method 
of  preparing  vegetable  tissue  for  paraffin  imbedding :  — 

"  Requisites :  (i)  Picro-corrosive  alcohol.  Heat  absolute 
alcohol  to  50°  C.,  saturate  with  picric  acid,  and  then  add 
bichlorid  of  mercury  to  saturation.  When  cool  decant.  This 
solution  may  be  made  in  quantity  and  kept.  (2)  Absolute  alcohol. 
(3)  Chloroform-alcohol,  chloroform,  and  absolute  alcohol  mixed 
in  equal  parts.  (4)  Chloroform.  (5)  Solid  paraffin,  melting 
point  46°-5o°  C.  (6)  Short,  wide-mouth  bottles.  (7)  Best  cork 
stoppers,  two  for  each  bottle ;  one  fitted  with  a  piece  of  glass 
tubing  i  cm.  in  diameter  and  3  cm.  long.  (8)  Number  of  glass 
rods  drawn  out  into  fine  points,  as  one  must  avoid  bringing 
metal  instruments  in  contact  with  the  picro-corrosive  fluid. 

"  Method :  A.  The  fixing  and  hardening  of  tissues :  Place 
the  tissue  in  at  least  fifty  times  its  bulk  of  the  picro-corrosive 
alcohol.  Leave  small  objects  (up  to  i  cubic  cm.)  for  twenty-four 
hours,  larger  objects  for  forty-eight  hours  and  upward,  in  the 
fluid.  Keep  the  bottle  well  corked. 

"  B.  The  replacement  of  the  picro-corrosive  alcohol  by  pure 
absolute  alcohol,  (i)  Pour  off  the  hardening  fluid  till  the  tissues 
are  just  covered.  Add  absolute  alcohol,  according  to  the  size 
of  the  tissue,  in  i-io  drops  every"  ten  minutes  till  the  tissue  is 
again  in  fifty  times  its  bulk  of  fluid.  After  each  addition  move 
the  bottle  very  gently  to  allow  the  added  alcohol  to  mix  with 
the  hardening  fluid.  Leave  the  tissue  in  this  diluted  mixture 
for  twenty-four  hours.  In  no  case  should  this  process  be  hur- 
ried, or  strong  diffusion  currents  will  be  set  up,  and  the  pro- 
toplasmic contents  of  the  cell  separate  from  the  cell  wall.  (2) 
Pour  off  the  fluid  till  the  tissue  is  just  covered,  and  add  absolute 
alcohol  to  the  original  bulk.  Move  about  the  bottle  every  three 
or  four  hours.  Most  of  the  picro-corrosive  material  will  thus  be 
extracted  after  twenty-four  hours.  (3)  Draw  the  fluid  off  rap- 
idly by  means  of  a  pipette,  and  add  absolute  alcohol  up  to  half  the 
original  bulk.  Any  drying  of  the  tissue  must  be  carefully  guarded 
against.  Leave  for  twenty-four  hours  and  repeat  the  process. 

1  Jour.  Roy.  Mic.  Soc.,  p.  686,  1891. 


88  BIOLOGICAL   LABORATORY   METHODS 

"  C.  The  replacement  of  the  alcohol  by  chloroform :  (i)  Pass, 
by  means  of  a  pipette,  the  chloroform-alcohol  mixture  to  the 
bottom  of  the  vessel,  when  the  tissue  will  float  on  the  mixture ; 
remove  then  the  superfluous  alcohol  by  a  pipette,  leaving  only 
enough  to  cover  the  tissue.  (2)  When  the  tissue  has  sunk  in  the 
chloroform-alcohol  mixture,  introduce  by  a  pipette  pure  chloro- 
form, on  which  the  tissue  will  float ;  the  fluid  above  the  tissue  is 
removed  by  a  pipette.  After  twenty-four  hours  the  tissue  may 
or  may  not  have  sunk  in  the  chloroform ;  if  not,  it  may  be  in- 
duced to  do  so  by  heating  the  chloroform  to  20°  C.  (not  higher)  ; 
if  this  fail,  a  little  sulphuric  ether  may  be  added.  After  the 
tissue  has  sunk,  leave  for  twenty-four  hours.  (3)  Place  a  fresh 
supply  of  chloroform  at  the  bottom  of  the  vessel  (fifty  times  the 
bulk  of  the  tissue),  and  if  there  is  a  distinct  line  of  demarcation 
between  the  newly  added  and  the  old  chloroform,  the  upper 
layer  should  be  removed  by  a  pipette. 

"  D.  The  replacement  of  the  chloroform  by  paraffin :  (i) 
Place  the  tissue  in  a  warm  chamber,  heated  to  25°  C. ;  add  solid 
paraffin  in  pieces  up  to  the  size  of  a  small  pea.  After  each  piece 
has  dissolved,  the  bottle  has  to  be  moved  about  very  gently  to 
hasten  the  mixing  of  the  paraffin,  which  will  be  in  the  upper 
layers,  with  the  chloroform.  Continue  until  no  more  paraffin 
dissolves.  Tissue  which  did  not  sink  in  pure  chloroform  will 
always  sink  as  soon  as  paraffin  is  added.  (2)  Place  the  tissue 
in  a  warm  chamber  heated  to  30°  C.  for  twenty-four  hours. 
(3)  Place  the  tissue  in  a  warm  chamber  heated  to  the  melt- 
ing point  of  the  paraffin  (46°  C.),  and  after  six  hours  replace 
the  ordinary  cork  stopper  (which  up  to  this  stage  has  always 
to  be  employed)  by  a  perforated  one.  This  method  is 
adapted  to  insure  a  gradual  giving  off  of  the  chloroform,  for 
I  find  that,  if  the  latter  be  driven  off  rapidly,  a  good  deal  of 
shrinkage  always  results.  When  all  the  chloroform  has  evapo- 
rated, —  i.e.  if  after  shaking  the  bottle  gently  one  is  unable  to 
detect,  by  smelling,  the  faintest  trace  of  chloroform,  —  then  the 
tissue  is  ready  for  sectioning.  (4)  The  tissues  should  not  be 
exposed  longer  than  just  necessary  to  the  temperature  of  melted 


IMBEDDING   METHODS  89 

paraffin,  but  should  be  imbedded  by  means  of  Leuckart's  type- 
metal  box." 

Herr  F.  Rosen  gives  the  following  method  for  imbedding  vege- 
table tissue  so  as  to  produce  the  least  contraction  possible : l  — 
"  The  objects,  thoroughly  dehydrated  in  absolute  alcohol,  are 
successively  transferred  (i)  to  a  mixture  of  equal  parts  of  abso- 
lute alcohol  and  bergamot  oil ;  (2)  to  pure  bergamot  oil ;  (3)  to 
a  mixture  of  equal  parts  of  bergamot  oil  and  paraffin ;  (4)  to 
paraffin  with  melting  point  of  45°  C.  ;  (5)  to  paraffin  with  melt- 
ing point  of  56°-58°  C.  for  twenty-four  hours. 

"  In  stages  (3)  and  (4)  the  fluids  should  be  kept  at  48° ;  in 
stage  (5),  at  60°.  In  order  to  make  the  sections  adhere  to  the 
slide,  they  are  placed,  while  still  in  paraffin,  on  a  drop  of  fluid 
which  can  be  evaporated  completely,  such  as  distilled  water,  or 
50  per  cent  pure  alcohol.  The  paraffin  is  dissolved  out  by 
xylol.  The  alcohol  must  be  completely  evaporated  before  the 
treatment  with  xylol,  otherwise  the  sections  will  get  free." 

Imbedding  in  celloidin  or  collodion.  —  This  method  is  adopted 
for  cutting  large  objects,  while  paraffin  is  used  for  small  objects. 
This  is  necessary,  because  if  paraffin  was  substituted  for  celloidin 
in  the  case  of  large  objects,  of  ten  millimeters  or  more,  perfect 
sections  would  not  be  secured,  since  the  paraffin  has  a  tendency 
to  split  when  the  knife  strikes  it.  On  the  other  hand,  celloidin 
will  not  yield  as  thin  sections  with  small  specimens  as  those 
produced  by  paraffin  and  is  not  suitable  therefore  for  minute 
anatomy. 

Celloidin  is  a  preparation  of  pure  pyroxylin  (nitrocellulose), 
manufactured  in  Germany  and  sent  out  in  the  form  of  tablets  of 
a  tough,  gelatinous  consistency.  It  is  soluble  in  ether  and  abso- 
lute alcohol.  Imbedding  in  collodion  was  first  introduced  by 
Duval,  in  1879,  an^  ^  was  afterward  improved  on  by  the  intro- 
duction of  celloidin.  The  method  recommended  in  the  use  of 
celloidin  is  as  follows  :  — 

The  object  is  first  thoroughly  dehydrated  in  absolute  alcohol, 
and  then  soaked  in  ether  until  saturated,  or  in  a  mixture  of  ether 
l  Jour.  Roy.  Mic,  •&?.,  p.  632,  October,  1894. 


90         BIOLOGICAL  LABORATORY  METHODS 

and  absolute  alcohol.  This  will  usually  require  several  days. 
From  this  solution  it  is  transferred  to  solutions  of  celloidin,  of 
various  degrees  of  consistency,  beginning  with  a  thin  solution 
and  passing  from  thicker  to  thicker  until  the  specimen  has  been 
thoroughly  impregnated  with  the  celloidin.  Busse  proposes 
using  three  solutions:  the  first  consisting  of  10  parts,  by 
weight,  of  celloidin  and  150  parts  of  ether  and  alcohol  mixture ; 
the  second,  consisting  of  10  parts  of  celloidin  and  105  parts  of 
the  ether-alcohol  mixture;  and  the  third  consisting  of  10  parts 
of  celloidin  and  80  parts  of  ether-alcohol  mixture.  The  object 
must  remain  in  the  first  bath  for  a  length  of  time  sufficient  to 
permit  of  thorough  penetration.  To  accomplish  this  may  require 
several  days.  After  soaking  in  the  last  solution,  the  specimen  is 
imbedded  in  the  celloidin  by  either  one  of  the  following  methods  : 
i.  The  imbedding  material  is  moulded  in  the  paper  box,  or 
metal  box,  mentioned  on  page  64.  The  bottom  of  the  box  is 
covered  with  a  layer  of  the  thick  celloidin  and  allowed  to  dry  by 
evaporation  ;  another  layer  is  placed  on  this  and  allowed  to  dry  ; 
and  so  on  until  a  sufficiently  thick  coating  is  secured  in  the 
bottom  of  the  box.  The  object  is  now  arranged  on  this  coating 
of  celloidin  and  fresh  solution  added  until  the  object  is  entirely 
covered  with  the  imbedding  mass  to  the  depth  desired.  After 
the  filling  has  been  completed,  the  box  is  placed  under  a  bell- 
glass  resting  on  a  ground-glass  plate  and  the  celloidin  allowed 
to  slowly  dry  in  the  alcohol  atmosphere  generated  from  the 
block  of  imbedding  material.  When  sufficiently  hard  to  resist 
the  pressure  from  the  ball  of  the  finger,  the  paper  or  metal  box 
is  removed  and  the  mass  is  immersed  in  chloroform,  where,  in  a 
short  time,  it  assumes  the  form  of  a  hard  coagulated  body 
resembling  wax  and  beautifully  transparent.  After  coming 
from  the  chloroform  it  is  placed  in  a  vessel  containing  white  oil 
of  thyme,  or  similar  oil,  where  it  will  assume,  in  a  few  hours,  the 
appearance  of  a  clear  glasslike  mass.1  It  is  best  to  use  the 
chloroform  immersion  for  small  objects,  but  for  larger  bodies  it 
is  recommended  by  many  microscopists  to  use  alcohol  of  85°.  In 

1  American  Naturalist,  XXVI,  p.  80,  1892. 


IMBEDDING   METHODS  9 1 

the  85°  alcohol  solution  the  block  of  imbedding  material  must 
remain  until  it  assumes  the  consistency  required  for  good  work 
in  the  microtome.  This  may  take  several  days.  The  vessel 
containing  the  alcohol  must  not  be  closely  corked.  After  the 
mass  of  celloidin  has  been  well  hardened,  it  can  be  preserved  for 
a  long  time  in  70°  alcohol. 

While  the  block  of  celloidin  is  being  cut,  it  must  be  kept 
moist  with  70°  alcohol,  since  it  dries  very  rapidly.  If,  however, 
the  oil  of  thyme  treatment  has  been  resorted  to,  the  knife  of  the 
microtome  may  be  lubricated  with  this  oil,  thus  making  it  pos- 
sible to  cut  thinner  sections.  Moreover,  the  oil  does  not  evapo- 
rate quickly  like  the  alcohol,  and  the  operation  may  be  stopped 
for  some  time  without  serious  detriment  to  the  celloidin  mass. 

If  the  object  has  not  been  stained  in  mass,  it  may  be  difficult 
to  distinguish  it  in  the  mass  of  transparent  celloidin.  Under 
this  condition  it  will  be  best  to  slightly  color  the  celloidin  with 
picric  acid  or  other  similar  stains. 

When  submitted  to  the  pressure  of  the  microtome  clamps  the 
celloidin  yields  slightly,  and  there  is  danger  of  distorting  the 
object  and  thus  destroying  its  histological  value.  To  avoid  this 
trouble,  the  block  of  celloidin,  after  being  properly  hardened,  is 
mounted  on  a  piece  of  wood  suitably  shaped  for  being  grasped 
by  the  microtome.  A  coat  of  the  thick  solution  of  celloidin  is 
spread  over  the  surface  of  the  wooden  block  and  allowed  to  dry. 
The  under  surface  of  the  celloidin  block  is  trimmed  to  expose 
fresh  surface  and  a  solution  of  the  celloidin  smeared  over  it. 
The  two  surfaces  thus  prepared  are  pressed  together,  and  then 
placed  in  a  solution  of  70°  alcohol  to  soak  for  a  few  hours,  in 
order  that  the  point  of  contact  may  become  hardened.  The 
wooden  block  is  now  clamped  in  the  microtome  and  the  sections 
cut  in  the  usual  way. 

2.  This  method  consists  in  building  up  the  block  of  celloidin 
on  the  wooden  support  from  the  start.  The  surface  of  the  wood 
is  slightly  roughened,  so  that  the  material  will  stick  securely, 
and  a  layer  of  the  thick  celloidin  is  spread  over  it  and  permitted 
to  dry,  and  then  another  layer  is  added,  and  the  process  repeated 


92         BIOLOGICAL  LABORATORY  METHODS 

until  the  foundation  is  built  up  high  enough,  when  the  object  is 
put  into  position  and  the  celloidin  placed  around  and  over  it 
until  it  is  entirely  covered  with  the  imbedding  mass.  The  hard- 
ening of  the  celloidin  and  after  treatment  are  the  same  as  used 
in  the  first  method. 

Herr  O.  Jelinek l  recommends  the  use  of  the  new  insulating 
material  called  stabilite  in  the  place  of  the  wooden  support  for 
celloidin  imbedding.  Its  advantage  consists  in  its  insolubility 
in  water  or  alcohol  (wood  and  cork  are  slightly  soluble  in  alcohol, 
giving  up  coloring  matters  and  tannic  acid)  ;  it  can  be  cut  by  a 
saw  into  any  shape  desired ;  it  is  firm  and  will  withstand  the 
pressure  of  the  microtome  ;  it  is  cheap  ;  it  may  be  readily  written 
on  with  pencil  or  ink,  which  are  not  easily  effaced  while  under 
treatment ;  it  is  not  hygroscopic  and  will  quickly  sink  in  alcohol, 
thus  bringing  the  imbedding  mass  in  thorough  contact  with  the 
hardening  solution  ;  it  is  not  attacked  by  dilute  hydrochloric  and 
sulphuric  acids. 

It  is  important,  says  Dr.  A.  Elsching,2  that  celloidin  solutions 
should  be  free  from  air  and  water.  To  effectually  deprive  cel- 
loidin of  water,*  the  tablets  should  be  cut  up  into  little  blocks, 
the  sides  of  which  are  not  bigger  than  5  mm.  These  are  placed 
between  folds  of  blotting-paper,  and  first  allowed  to  dry  at  the 
room  temperature,  and  then  desiccated  in  an  incubator.  At  this 
stage  they  should  be  of  a  yellowish  hue  and  of  horny  consistence. 
Absolute  alcohol  may  be  easily  obtained  entirely  free  of  water, 
by  repeated  treating  with  freshly  dried  coppef  sulphate.  The 
dried  cubes  are  placed  in  a  narrow-necked  bottle,  with  air-tight 
stopper,  and  form  a  layer  not  exceeding  one-fourth  the  volume 
of  the  bottle.  The  celloidin  is  then  just  covered  with  the  abso- 
lute alcohol,  and  allowed  to  stand  for  about  twenty-four  hours, 
after  which  the  ether  is  poured  in.  In  a  very  short  time  the 
celloidin  is  all  dissolved,  and  thus,  as  no  stirring  is  required, 
the  solution  is  kept  free  from  air  bubbles. 

1  Zeitschr.  f.  wiss.  Mikr.,  XT,  p.  237,  1894. 

zjour.  Roy.  Mic.  Soc.,  p.  404,  June,  1894.  And  Zeitschr. /.  wiss.  Mikr.,  pp.  445- 
446,  1893. 


IMBEDDING   METHODS  93 

Freezing.  —  Freezing  in  the  place  of  imbedding  is  often  resorted 
to  by  microscopists  in  cutting  sections  from  soft  tissues,  and  ex- 
cellent results  are  generally  secured.  The  specimen  is  fastened 
to  the  plate  of  the  microtome  by  a  gum,  which  is  also  frozen  and 
firmly  holds  the  specimen  during  the  cutting  of  sections.  In 
using  this  method,  care  must  be  exercised  to  prevent,  as  far  as 
possible,  the  formation  of  crystals  in  the  cells  of  the  delicate 
tissues,  so  that  rupture  will  not  take  place.  This  trouble  is 
greatly  overcome  by  the  use  of  some  gummy  substance  which 
will  not  form  crystals  when  the  specimen  is  frozen.  The  gum 
is  infiltrated  into  the  specimen.  It  is  recommended  in  the 
Journal  of  the  Royal  Microscopical  Society  that,  when  the  speci- 
men is  soaked  in  oil  of  aniseed  for  some  hours  —  twelve  to 
twenty-four  —  the  freezing  may  be  safely  accomplished  without 
detriment  to  the  tissues.  The  oil  is  dissolved  out  of  the  sections 
by  the  use  of  alcohol.  The  freezing  is  successfully  done  by  the 
apparatus  illustrated  and  described  on  pages  94  to  98. 

Freezing  microtomes  for  use  with  carbon-dioxid  tanks.  —  (Con- 
densed from  an  article  written  by  Professor  C.  R.  Bardeen,  of 
Johns  Hopkins  University,  for  the  Journal  of  Applied  Microscopy, 
Vol.  IV,  p.  1320.) 

Of  late  carbon  dioxid  has  been  much  utilized,  especially  by 
pathologists,  as  a  means  of  freezing  tissues  for  sectioning.  The 
convenience  with  which  fluid  carbon-dioxid  may  be  obtained  in 
tanks,  and  its  power  of  rapid  freezing,  have  caused  it  to  be  pre- 
ferred to  ether  and  similar  fluids.  In  every  active  pathological 
laboratory  the  freezing  microtome  is  in  daily  use.  Perhaps  its 
greatest  value  lies  in  the  fact  that  thin  sections  may  be  made 
within  a  few  minutes  after  the  removal  of  the  tissue  from  the 
body,  and  in  a  few  minutes  more  these  sections  may  be  hardened, 
stained,  cleared,  and  mounted.  The  surgeon  may  thus  be  given 
a  positive  diagnosis  of  the  microscopic  condition  of  the  diseased 
tissues  while  he  proceeds  with  the  operation. 

The  carbon-dioxid  microtomes  commonly  used  have,  however, 
several  drawbacks,  which  have  served  to  render  them  far  less 
useful  than  they  should  be.  From  the  practical  standpoint  their 


94 


BIOLOGICAL  LABORATORY  METHODS 


most  serious  drawbacks  are  a  tendency  to  become  clogged  and 
a  great  wastefulness  of  gas.  From  a  scientific  standpoint  lack 
of  control  over  the  temperature  of  the  freezing  stage  serves  to 
give  rise  to  an  "  over-freezing,"  which  produces  marked  altera- 
tions in  the  tissues.  In  order  to  remedy  these  defects  the 
machine  described  below  was  devised. 


FIG.  57.  —  Bardeen's  Freezing  Microtome. 


Figure  57  shows  the  machine  as  it  stands  ready  for  use.  It 
is  supported  directly  by  the  nozzle  of  the  carbon-dioxid  tank. 
This  offers  a  firm  and  convenient  means  of  attachment,  but, 
if  desired,  a  heavy  tubing  may  be  utilized  to  connect  tank  and 
machine.  When  the  microtome  is  screwed  directly  upon  the 
carbon-dioxid  tank,  it  is  necessary  that  the  latter  lie  in  a  hori- 
zontal position.  On  the  other  hand,  if  an  L-shaped  piece  of 


IMBEDDING   METHODS 


95 


tubing  be  utilized  to  connect  freezing  microtome  and  tank,  the 
tank  may  be  placed  at  any  desired  angle. 

The  valve  of  the  tank  is  used  to  control  the  escape  of  gas  into 
the  machine. 

The  axis  and  main  support  of  the  instrument  consists  of  a 
stout  tube  with  a  narrow  lumen  (K-D,  Fig.  58).  This  axial  tube 
is  united  by  a  nut  (/,  Figs. 
57  and  58),  either  directly 
to  the  nozzle  of  the  tank, 
or,  in  case  a  connecting 
tube  is  used,  to  the  latter. 

On  the  top  of  the  axial 
tube  the  freezing  stage  (A, 
Fig.  57;  A-C,  Fig.  58)  is 
screwed.  This  stage  piece 
consists  of  two  parts,  a  base 
and  a  cover.  The  base  is 
the  part  screwed  into  the 
upper  end  of  the  axial  tube 
(C,  Fig.  58).  To  this  base 
the  cover  piece  is  screwed 
(A,  Fig.  58).  Between  the 
base  of  the  stage  and  the 
axial  tube  is  placed  a  thin 
brass  plate  (D,  Fig.  58), 
with  a  very  narrow  aperture 
at  its  centre.  Through  this 
narrow  aperture  the  carbon 
dioxid  escapes  into  the 
lumen  of  the  stage  piece  (C,  Fig.  58).  The  difference  in  press- 
ure on  the  two  sides  of  the  brass  plate  causes  a  very  rapid  ex- 
pansion of  gas  between  the  cover  and  base  of  the  freezing  stage. 
The  passage  open  for  the  escape  of  gas  from  the  lumen  of  the 
base  (C,  Fig.  58)  to  the  external  world  is  in  the  form  of  a  spiral 
passage,  which  finally  opens  out  through  the  side  of  the  cover, 
as  shown  in  Fig.  57,  A.  Between  the  cover  and  base  of  the 


FIG.  58.  — Section  of  Bardeen's  Freezing 
Microtome. 


96        BIOLOGICAL  LABORATORY  METHODS 

freezing  stage  an  asbestos  washer  is  placed.  The  expanding 
gas,  therefore,  can  absorb  little  heat  from  the  base  of  the  stage. 
Almost  all  heat  absorption  must  take  place  from  the  cover. 
This  heat  absorption  is  greatly  facilitated  by  the  metallic  spiral, 
which  projects  down  from  the  cover  so  as  to  give  rise  to  the 
spiral  passage  through  which  the  gas  escapes. 

Through  the  mechanism  here  described,  far  the  greater  part 
of  the  heat-absorbing  power  of  the  expanding  gas  is  utilized  to 
lower  the  temperature  of  the  surface  of  the  cover  of  the  freezing 
stage.  The  temperature  of  the  rest  of  the  machine  is  but  little 
altered.  Good  control  of  the  temperature  of  the  freezing  stage 
can  be  thus  maintained.  This  control  is  further  rendered  pos- 
sible by  the  valve  of  the  tank.  If  this  valve  be  turned  on  full, 
the  temperature  of  the  cover  of  the  freezing  stage  will  be  quickly 
reduced  to  a  very  low  point.  Tissue  placed  on  it  is  quickly 
frozen.  On  the  other  hand,  if  the  gas  is  not  permitted  to  escape 
from  the  tank  with  full  force,  the  difference  in  pressure  on  the 
two  sides  of  the  brass  plate  is  less  and  heat  absorption  from  the 
cover  is  less  marked.  In  this  way  tissues  placed  on  the  cover 
may  be  slowly  frozen  without  subjecting  them  to  severe  cold. 
Thus,  too,  a  constant  low  temperature  may  be  maintained  by 
opening  the  tank  valve  to  the  required  point. 

The  mechanism  for  controlling  the  thickness  of  the  sections 
is  equally  simple.  On  the  lower  end  of  the  axial  tube  a  movable 
wheel  (/,  Figs.  57  and  58)  is  placed.  This  wheel  moves  up  and 
down  the  axial  tube  on  a  screw-thread,  cut  twenty-five  threads 
to  the  inch.  A  complete  revolution  of  the  wheel,  therefore, 
raises  or  lowers  it  a  millimeter.  The  margin  of  the  wheel  is 
divided  into  fifty  spaces,  each  of  which  therefore  represents 
twenty  microns.  A  pointer  (TV,  Fig.  57)  serves  to  indicate  the 
number  of  spaces  passed  in  a  partial  revolution  of  the  wheel, 
and  thus  to  show  the  thickness  of  the  sections  cut. 

The  knife  stage  (F—B,  Figs.  57  and  58)  consists  of  a  tubal 
base  (/?),  which  surrounds  an  axial  tube  and  rests  on  the  mov- 
able wheel ;  and  of  two  flanges  (B),  which  extend  above  the 
freezing  stage  on  each  side  for  the  support  of  the  cutting  blade. 


IMBEDDING   METHODS 


97 


The  base  of  the  knife  stage  is  moved  up  the  axial  tube  by  screw- 
ing the  wheel  upward.  It  is  forced  down  the  axial  tube  by  the 
spring  (£,  Figs.  57  and  58)  whenever  the  wheel  is  turned  so 
as  to  be  carried  downward.  The  flanges  of  the  knife  stage 
support  parallel  glass  tracks,  upon  which  the  cutting  blade  is  car- 
ried to  and  fro. 

For  cutting  sections  a  razor  or  a  plane,  or  almost  any  good 
steel  blade  with  a  straight  edge,  may  be  used. 


Jung's  Freezing  and  Paraffine 
Microtome,  with  Automatic 
Feed  Attachment, 

FIG.  59. 

Jung's  freezing  and  paraffin  microtome. — This  instrument  is 
used  by  workers  in  this  country  and  in  Europe.  It  has  a  freez- 
ing and  automatic  feeding  attachment,  and  is  adjustable  to  any 
desired  thickness  from  five  microns  upward.  The  knife  m  is 
clamped  to  the  vertical  shaft  a  by  means  of  the  screw  e.  The 
lever  k  moves  the  knife  over  the  object  o.  The  illustration  (Fig. 
59)  shows  the  freezing  attachment  in  place.  By  pressing  the 
rubber  bulb  the  carbon  dioxid  is  transmitted  from  the  bottle  h 


98  BIOLOGICAL  LABORATORY   METHODS 

into  the  cylinder  c,  and  by  rapid  evaporation  the  heat  is  extracted 
from  the  object,  which  soon  freezes  to  the  proper  consistency 
for  cutting  into  sections.  This  freezing  attachment  may  be 
separated  from  the  microtome  and  the  cylinder  <*  substituted  if 
sections  are  to  be  made  of  objects  not  requiring  freezing. 

Figure  60  illustrates  the  apparatus  which  is  applicable  to  the 
microtomes  shown  in  Figs.  30  to  35,  when  freezing  is  resorted 
to  for  cutting  sections.  The  attachment  A  is  a  cylindrical  box 
upon  which  the  specimen  to  be  frozen  is  placed.  The  spray  of 


FIG.  60.  —  Ether  or  Rhizolene  Freezing  Attachment. 

ether  or  rhizolene  is  projected  against  this  stage  by  means  of 
the  atomizer  C.  The  excess  of  fluid  is  caught  by  the  bottle  D, 
Orienting.  —  In  orientation  the  sections  are  made  in  definite 
planes  in  relation  to  certain  points  so  that  the  operator  is  able  to 
determine  the  exact  plane,  the  direction  the  microtome  knife 
has  travelled,  and  the  precise  thickness  of  the  sections.  If  it 
is  desired  to  reconstruct  the  object  which  has  been  sectioned, 
it  may  be  so  accomplished  if  the  orientation  has  been  carefully 
done.  There  are  several  methods  for  orientation  adopted  by 
microscopists,  but  the  description  of  two  will  suffice  in  this  con- 
nection to  give  the  student  a  conception  of  what  the  general 
method  is. 


IMBEDDING   METHODS  99 

Woodworth's  method  for  orienting  small  objects.  —  In  pre- 
paring small  objects  for  the  microtome,  Mr.  W.  McM.  Woodworth, 
of  Harvard  University,  found  some  difficulty  in  orienting  the 
specimens  so  that  he  could  obtain  sections  in  definite  planes, 
until  he  developed  the  following  plan.  He  took  paper  with 
raised  parallel  lines,  or  a  grained  or  rep  surface,  such  as  is  usual 
with  linen  cloth  writing  papep,  and  cut  rectangular  strips  5X15 
mm.,  so  that  the  cut  edges  were  parallel  and  perpendicular  to 
the  raised  lines  on  the  surface  of  the  paper.  He  glued  the 
smooth  side  of  the  paper  to  a  slip  of  glass  by  means  of  gum 
arabic ;  after  drying,  the  exposed  or  line  side  of  the  paper  was 
covered  with  a  thin  coating  of  gum  arabic  solution,  applied  with 
a  brush.  And  after  this  second  coat  dried,  he  placed  on  it  a  thin 
coating  of  celloidin,  made  of  flexible  collodion  diluted  with  three 
parts  of  ether.  Apply  this  collodion  just  before  using  the  mount 
for  orienting  the  object,  because  if  applied  some  time  before 
using  it  will  crack  and  fall  off.  The  object  to  be  oriented,  pre- 
viously soaked  in  turpentine,  is  drained  of  the  surplus  turpentine 
by  means  of  blotting-paper,  and  then  arranged  on  the  surface  of 
the  collodionized  paper,  where  it  will  stick  if  the  oil  has  been 
well  drained  off.  The  axes  of  the  object  are  arranged  with  the 
desired  relation  to  the  raised  lines  on  the  paper.  When  thus 
suitably  oriented  the  glass  slide  is  exposed  under  a  bell-glass  to 
the  ether  vapor  for  a  few  seconds ;  this  will  soften  the  collodion,  to 
which,  when  it  dries  again,  the  object  will  be  found  firmly  attached 
in  the  desired  position.  Now  put  a  drop  of  turpentine  on  the 
object,  and  the  mount  is  ready  for  the  paraffin  bath.  After  soak- 
ing for  a  time  in  the  paraffin,  the  metal  box  is  placed  around 
the  glass  slip,  and  the  object  is  imbedded  in  paraffin.  When  the 
paraffin  cools,  carefully  remove  the  metal  box  and  trim  the  block 
so  that  the  edges  of  the  paper  show  all  around.  Place  in  a  vessel 
of  water,  and  the  gum  arabic  will  be  dissolved,  and  the  paper  may 
be  stripped  off,  leaving  the  paraffin  block  marked  with  the  raised 
lines,  and  the  object  resting  just  below  them  with  the  collodion 
film  between  the  object  and  the  delicate  lines.  These  lines  will 
now  permit  the  correct  orientation  of  the  block  in  the  microtome 


IOO  BIOLOGICAL  LABORATORY   METHODS 

and  render  it  possible  to  section  the  object  along  certain  known 
planes.  (Bull.  Mus.  Comp.  Zool.,  XXV,  pp.  45-47.) 

Eyclesheimer's  method  of  orienting.  — The  metal  box  used  for 
imbedding  is  perforated  with  a  number  of  small  holes  along  the 
sides  and  ends  ;  through  these  are  run  silk  threads,  and  fastened 
on  the  outsides  of  the  box  by  means  of  celloidin.  The  object  is 
oriented  on  these  silk  threads,  and  the  imbedding  mass  is  poured 
in  and  hardened.  After  the  hardening  has  been  completed,  the 
ends  of  the  threads  are  detached  by  a  drop  of  ether,  and  the 
ends  hanging  on  one  side  and  on  one  end  of  the  box  are  black- 
ened with  lampblack  mixed  with  celloidin.  The  other  ends  of 
the  threads  are  now  pulled  through  the  box,  and  the  lampblack 
leaves  distinct  lines  through  the  imbedding  mass  which  serve  to 
orient  the  block  and  its  object  on  the  microtome. 

Fixing  the  sections  to  the  glass  slips.  —  After  the  sections 
have  been  properly  cut,  with  or  without  the  imbedding  mass, 
described  in  previous  pages,  and  it  is  proposed  to  mount  them 
on  glass  slips  for  permanent  preservation,  it  is  necessary  to 
resort  to  some  method  for  fastening  or  fixing  them  on  the  glass, 
so  that  they  will  not  change  their  positions  during  the  treatment 
in  staining  and  in  mounting. 

If  the  specimen  was  stained  in  bulk,  then  mounted  or  imbedded 
in  paraffin  and  the  sections  cut  with  the  imbedding  mass  attached, 
the  following  methods  are  recommended  as  giving  the  most 
satisfactory  results  in  causing  the  sections  to  adhere  to  the 
glass  slip  while  the  imbedding  mass  is  being  removed,  and  during 
the  progress  of  other  manipulations  necessary  to  complete  the 
mount :  — 

MAYER'S  ALBUMEN  FIXATIVE  :  — 

White  of  egg 50  cc. 

Glycerin 50  cc. 

Salicylate  of  soda I  gramme 

The  method  for  using  this  fixative  is  as  follows  :  A  small  drop 
of  the  albumen  is  placed  on  the  slip,  or  the  cover-glass,  and,  by 
means  of  a  clean  finger,  spread  over  the  place  where  the  section  is 


IMBEDDING  METHODS  IOI 

to  be  mounted  until  a  very  thin  film  of  the  albumen  is  secured,  — 
the  thinner  the  better.  Arrange  the  sections  in  the  order  in  which 
they  are  to  remain,  and  place  the  glass  slip  under  a  bell-glass  for 
several  hours  until  the  water  has  evaporated.  Warm  for  a  few 
minutes  in  the  water-bath,  and  as  the  paraffin  is  melted  the 
albumen  is  carried  away  with  it  and  the  sections  are  left  firmly 
adhering  to  the  glass.  The  tissues  may  now  be  .stained  and 
dehydrated  without  danger  of  displacing  the  sections.  The 
author  in  using  this  method  prefers  to  attach  the  sections  to  the 
cover-glass,  because  so  little  space  is  occupied  by  the  cover- 
glasses  ;  and,  if  the  slips  have  rings  spun  on  them,  there  is  diffi- 
culty of  destroying  these  rings  while  staining  and  dehydrating 
the  sections  after  they  are  attached  to  the  glass  slip. 

If  the  sections  are  found  to  be  inclined  to  fold,  the  method  of 
procedure  is  as  follows  :  After  preparing  the  glass  with  the  albu- 
men mixture,  place  on  it  a  drop  of  water  and  spread  over  the 
surface,  arrange  the  sections,  and  warm  the  slide  until  the  sec- 
tions open  and  flatten  out.  Evaporate  the  water  at  a  tempera- 
ture below  the  melting  point  of  paraffin,  then  melt  the  paraffin 
and  transfer  to  a  vessel  containing  benzol  or  xylol,  which  will 
dissolve  all  the  paraffin  remaining.  This  will  generally  take  an 
hour  or  two. 

Huber's  method  (Jour.  App.  Mic.,  Vol.  I,  p.  103).  This 
method  is  conducted  with  the  sections  on  the  cover-glass. 
These  glasses  must  be  carefully  cleaned  before  they  are  used 
for  this  purpose.  This  is  accomplished  by  placing  them  in 
sulphuric  acid,  rinsing,  transferring  to  vessel  containing  acetic 
acid,  and  after  remaining  in  this  acid  a  few  minutes  the  cover- 
glasses  are  rinsed  again  and  then  placed  in  a  vessel  containing 
alcohol.  Wipe  them  dry  with  a  clean  soft  cloth,  and  they  are 
ready  for  the  process.  Heat  a  porcelain  dish  containing  water, 
with  several  of  the  paraffin  sections  floating  on  the  water,  until 
the  sections  just  begin  to  unfold,  remove  the  flame,  taking  care 
not  to  heat  sufficiently  to  cause  the  paraffin  to  melt.  When  these 
sections  are  flattened  out,  place  the  others  to  be  mounted  in  the 
water  until  they  also  are  flattened  out.  In  the  meantime  coat 


IO2  BIOLOGICAL   LABORATORY   METHODS 

the  cover-glasses  with  the  albumen  fixative  as  directed  above. 
Grasp  one  of  these  glasses  in  the  forceps  and  pass  it  under  one 
of  the  flattened-out  sections  on  the  water,  gently  lift  the  glass  up 
under  the  section  until  it  comes  in  contact  with  the  albumen 
surface,  and  then  with  the  assistance  of  the  section  lifter,  or  a 
needle,  hold  the  section  down  in  contact  with  the  surface  of  the 
glass  and  draw  from  the  water.  Drain  the  water  off  by  means  of 
filter-paper  or  a  blotter.  Place  the  cover-glass  aside  until  the 
water  evaporates,  from  five  to  eight  hours ;  or  heat  in  the  water- 
bath  with  a  temperature  not  above  40°  C.  After  the  water  has 
evaporated  hold  the  cover-glass  over  a  flame  until  the  paraffin  is 
melted,  care  being  taken  not  to  overheat,  and  then  transfer  to  a 
vessel  containing  xylol  until  all  of  the  paraffin  is  dissolved,  which 
will  take  two  or  three  minutes.  Drain  off  the  xylol  and  treat 
sections  with  alcohol  to  remove  the  xylol,  and  stain  or  clarify  and 
mount  in  balsam. 

Eisen's  method  (Zeitsckr.f.  wiss.  Mikr.,  Bd.  XVI)  :  — 
The  basis  of  this  method  is  alcohol  in  the  place  of  the  albu- 
men given  in  the  above  methods.  The  slide  is  flooded  with  70 
to  85  per  cent  alcohol,  and  the  sections  are  arranged  on  it. 
Hold  over  a  flame  until  the  sections  flatten  out,  but  taking  care 
not  to  melt  the  paraffin.  Drain  off  the  alcohol  by  means  of  filter- 
paper  or  blotter.  Place  on  the  slip  a  piece  of  smooth  blotting- 
paper,  the  size  of  the  glass,  which  has  been  saturated  with  alcohol 
the  same  strength  as  that  used  for  flattening  out  the  sections. 
Put  on  top  of  this  blotter  another  dry  blotter,  and  press  them 
well  in  contact  with  the  glass.  The  object  of  this  is  to  cause 
the  sections  to  adhere  in  close  contact  with  the  glass.  Remove 
the  blotters,  take  off  all  lint,  and  dry  at  a  temperature  not  above 
40°  C.  Dissolve  the  paraffin  as  given  in  former  methods.  It 
is  claimed  that  this  method  will  cause  the  sections  to  attach 
firmly  to  the  glass,  and  the  time  consumed  in  completing  the  work 
is  much  shorter  than  that  required  in  the  albumen  process. 
Koninski's  method  (Zeitschr.f.wiss.Mikr.,  XV,  p.  161,  1898): 

1.  Cover  slide  with  film  of  gelatin. 

2.  When  gelatin  has  set,  arrange  the  sections. 


IMBEDDING   METHODS 


103 


3.  Smooth  down  with  warm  water. 

4.  Warm  plate  until  gelatin  melts,  and  drain  off  surplus  gela- 

tin with  blotter. 

5.  When  dry,  place  in  pure  formalin  for  ten  minutes. 

The  section  is  now  so  firmly  attached  that  "  boiling  water  will 
lot  remove  it." 

The  water  method  for  fixing  sections  to  the  slip.  —  The  principle 
involved  in  this  method  is  the  molecular  attraction  between  the 
rlass  and  the  section.  The  operation  is  performed  as  follows : 
Clean  the  glass  slip  perfectly  by  first  immersing  it  in  alcohol  for 
>me  time  and  then  rinsing  it  with  water.  Rub  the  glass  thor- 
mghly  with  a  clean  cloth  until  a  film  of  water  will  stand  over  the 
surface  uniformly  and  not  in  separate  drops.  If  rubbing  with 
te  cloth  will  not  accomplish  this,  then  apply  with  the  cloth  some 
inely  divided  chalk  and  rub  again,  wash  with  water  and  dry,  and 
if  the  film  still  refuses  to  stand  evenly  over  the  surface,  reject 
glass  and  try  another.  The  cleaning  is  very  important  to  a 
successful  completion  of  the  fixing.  The  sections  cut  in  paraf- 
in,  if  inclined  to  roll  or  bend,  are  placed  on  the  surface  of  warm 
water  until  they  unroll,  but  care  must  be  exercised  not  to  heat 
enough  to  cause  the  paraffin  to  melt.  Spread  over  the  glass 
slip  a  thin  film  of  water  and  place  the  sections  on  it,  apply  a 
gentle  heat,  below  the  melting  point  of  the  paraffin,  until  the 
water  is  entirely  dried  out,  and  the  sections  will  be  left  in  close 
contact  with  the  glass,  so  that  the  after  treatment  with  the  stain- 
ing and  other  solutions  will  not  remove  it.  The  paraffin  may  be 
removed  by  dissolving  in  a  tube  containing  xylol.  Do  not  place 
the  glass  slips  containing  the  sections  in  solutions  which  are 
alkaline  in  reaction,  because  alkaline  fluids  will  detach  the  sec- 
ions.  To  be  successful  with  this  method  there  must  be  a  con- 
tinuous surface  in  contact  with  the  glass,  and  the  surface  of  the 
glass  must  be  well  cleaned.  The  larger  the  sections  and  the 
thinner  they  are,  the  more  perfect  will  be  the  results. 

Sections  which  are  cut  in  celloidin  or  collodion  imbedding 
masses  must  be  fixed  to  the  slip  by  means  of  Summer's  ether- 
vapor  method.  The  sections  are  first  placed  in  95  per  cent 


104  BIOLOGICAL  LABORATORY   METHODS 

alcohol  for  a  few  minutes,  and  then  arranged  on  the  slip ;  sul- 
phuric ether  vapor  is  poured  on  the  slip  from  a  bottle  half  full  of 
the  liquid  ether.  The  celloidin  will  become  softened  and  trans- 
parent. The  slip  is  now  transferred  to  a  vessel  containing  95 
per  cent  alcohol,  when  it  will  be  found  that  the  sections  are 
firmly  fastened  to  the  glass.1 

Weigert's  collodion  method  (Lee's  "  Vade-Mecum,"  p.  221; 
also  Zeitschr.f.  wiss.  Mikr.,  p.  490,  1885):  — 

Wet  the  knife  with  alcohol  during  the  cutting  of  sections. 
Soak  a  piece  of  porous  paper  with  alcohol  and  bring  it  in  contact 
with  the  section  on  the  knife,  and  then  by  a  horizontal,  upward 
motion  detach  the  section  from  the  knife  ;  it  will  remain  in  contact 
with  the  paper  if  carefully  worked.  Collodionize  the  glass  slips 
by  pouring  on  them  collodion  and  spreading  to  a  thin  film.  Take 
one  of  these  collodionized  slips  and  bring  the  paper  in  contact 
with  it,  with  the  section  down  on  the  surface  of  the  glass ;  press 
gently  until  the  section  is  brought  in  connection  with  the  glass 
and  then  remove  the  paper.  Remove  any  excess  of  alcohol  by 
means  of  blotting-paper,  and  cover  the  plate  with  collodion  and 
place  in  eighty  per  cent  alcohol  until  wanted  for  staining  or  other 
manipulation. 

1  American  Monthly  Microscopical  Journal,  p.  73,  1887. 


CHAPTER   VI 

STAINS,    THEIR    PREPARATION    AND    USE 

Stains.  — The  staining  of  tissue  is  resorted  to  in  order  that  the 
groups  of  cells  in  the  object  under  examination  may  be  clearly 
marked  and  thus  become  distinctive.  Without  this  method  of 
staining  the  sections  it  would  be  very  difficult,  in  some  instances, 
to  distinguish  the  minute  part  sought  for  from  the  rest  of  the 
tissue  surrounding  it. 

The  requisites  for  a  good  stain  are  :  — 

1 .  When  in  full  strength  its  action  should  be  intense. 

2.  It  should  have  selective  properties  rather  than  diffusive. 

3.  It  should  penetrate  well  all  portions  of  the  specimen,  stain- 
ing equally  the  similar  tissue  near  the  surface  and  toward  the 
centre  of  the  section. 

4.  The  color  should  be  permanent. 

5.  It  should  not  disintegrate  the  specimen,  even  though  the 
process  be  prolonged. 

All  stains  may  be  divided  into  two  groups  :  — 

1.  General  stains. 

2.  Selective  stains. 

(a)  Nuclear  stains. 

(b)  Cytoplasm  or  nucleoplasm  stains. 

(c)  Cell-development  stains. 

General  stains  act  on  all  parts  of  the  subject  alike,  while  selec- 
tive stains  pick  out  one  or  more  groups  of  fibre  in  the  section, 
and,  when  properly  applied,  leave  all  other  systems  of  the  section 
unaffected. 

To  produce  the  best  results  with  the  selective  stains,  two 
methods  have  been  proposed  :  — 

i.    The  staining  must  be  stopped  just  as  soon  as  the  parts  to 


IO6       BIOLOGICAL  LABORATORY  METHODS 

be  colored  have  received  the  maximum  degree  of  dye  and  the 
rest  of  the  section  is  beginning  to  be  affected. 

2.  The  section  may  be  left  in  the  selective  stain  and  continued 
much  beyond  the  first  step,  and  the  extra  color  eliminated  by 
washing  in  alcohol,  or  dilute  hydrochloric  acid,  in  which  the 
color  stain  is  soluble.  This  is  known  as  Flemming's  method, 
and  it  is  only  applicable  to  staining  sections  and  must  not  be 
used  for  staining  in  toto. 

Ehrlich  divides  the  anilin  dyes  into :  — 

1.  Acid. 

2.  Basic. 

The  first  is  subdivided  into : 

1.  Eosin  and  fluorescin. 

2.  Picric  acid  and  aurantia. 

3.  Tropaeolin. 

4.  Alizarin  purpurin,  rosol  acid. 

The  second  group  is  subdivided  into :  — 

Fuchsin,  methyl-violet,  gentian  violet,  methylene-blue,  vesuvin, 
Bismarck  brown. 

The  following  single  stains  are  recommended  for  general  use 
in  the  laboratory  :  — 

Anilin-green.  —  This  is  a  delicate  nuclear  stain  and  stains  the 
nucleoli  in  brilliant  color.  Stain  in  saturated  solutions  and 
wash  out  with  strong  alcohol.  Clear  with  cedar  or  bergamot 
oil. 

Bismarck  brown.  —  This  is  also  a  nuclear  stain.  Dissolve  in 
alcohol.  This  stain  is  especially  valuable  for  mass-staining, 
and  although  it  acts  very  rapidly,  it  never  overstains.  It  is 
permanent.  For  protoplasm,  connective  tissue,  bacteria,  and 
living  organisms  Bismarck  brown  gives  good,  results.  If  used  in 
staining  sections,  the  indirect  method  should  be  adopted.  Wash 
after  staining  in  absolute  alcohol. 

Carmine.  —  There  are  a  number  of  carmine  solutions  differing 
in  the  other  ingredients  combined  with  the  carmine.  Such,  for 
instance,  as  ammonia-carmine  (Gerlach's  formula),  alum-car- 
mine (Grenadier's  formula),  borax-carmine  (Grenacher's  for- 


STAINS,  THEIR   PREPARATION  AND   USE  IO/ 

mula),  picro-carmine  (Ranvier's  formula),  Mayer's  carmalum,  or 
alum-carmine.  Carmalum  is  one  of  the  very  best  of  the  car- 
mine dyes  for  staining  bacteria,  and  for  other  tissues,  and  it 
is  therefore  given  with  a  strong  recommendation  for  those 
purposes  for  which  it  was  developed.  The  formula  is  as 
follows :  — 

Carminic  acid I  gramme 

Alum 10  grammes 

Distilled  water 200  cc. 

Dissolve  with  heat  and  filter. 

Add  a  few  crystals  of  thymol  to  preserve  the  stain. 

After  staining  wash  out  with  water. 

Dahlia  or  Hoffman's  violet.  —  Dissolve  in  water  and  use  either 
neutral  or  acidified  with  acetic  acid.  Wash  out  with  strong 
alcohol.  Professor  A.  B.  Aubert  ("  The  Microscope,"  XI,  p.  270) 
recommends  the  following  formula,  which  is  suitable  for  nuclear 
and  fresh  tissue  stains  :  — 

Glacial  acetic  acid 12.5  cc. 

Absolute  alcohol 50     cc. 

Water 100     cc. 

Dahlia  nearly  to  saturation. 

This  formula  will  stain  in  twelve  hours  or  less. 

Eosin.  —  Stains  protoplasm  deeply,  muscles,  nuclei.  Also 
sieve  tubes.  This  stain  is  especially  valuable  as  a  contrast  stain  ; 
for  this  purpose,  see  compound  staining. 

Gentian  violet.  —  This  stain  is  one  of  the  most  important 
single  stains  and  is  also  much  used  in  compound  staining.  It 
is  especially  valuable  for  staining  living  tissue  and  has  the 
property  of  coloring  living  cells  without  destroying  their  vitality. 
It  is  soluble  in  either  water  or  in  alcohol.  Ehrlich's  formula  is 
probably  the  best  form  for  this  stain  :  — 

Gentian  violet    .......         I  gramme 

Anilin  oil 3  grammes 

AlcohoJ      .         . 15  grammes 

Water 80  grammes 


IO8  BIOLOGICAL  LABORATORY  METHODS 

Tissues  will  stain  in  this  within  twenty-four  hours,  at  ordinary 
temperatures,  but  by  raising  the  temperature  the  time  may  be 
much  shortened.  After  staining  wash  in  alcohol,  then  place  in 
chromic  acid,  and  then  again  in  alcohol  to  dehydrate,  and 
clarify  in  oil  of  cloves,  and  mount  in  balsam. 

This  stain  is  useful  for  bacteria,  decolorized  chlorophyl 
bodies. 

Methyl-green.  —  Dissolve  in  water  and  add  a  little  acetic  acid 
(0.75  per  cent).  A  standard  stain,  and  is  valuable  for  coloring 
nuclei  of  fresh  tissue  and  nerves.  Wash  with  water  slightly 
acidified.  Complete  fixation  is  accomplished  by  the  after  use  of 
the  vapor  of  osmium  and  Ripart's  and  Petit's  solution,  viz.:  — 

Copper  chloride     .  .         ...  .  -  .  '.  0.39  grammes 

Copper  acetate      ,  .         ,  .  .  .  0.30  grammes 

Acetic  acid  crystals  .         »  .  .  .  I       gramme 

Camphor  water      .  .  •   .  .  .  .  75       grammes 

Distilled  water       .  .         .  .  •  •  75       grammes 

The  action  of  the  osmium  is  to  strengthen  the  fixing  proper- 
ties of  the  solution. 

Safranin.  —  Dissolve  in  water  and  alcohol,   viz.:  — 

Safranin  .         .    •     ;         .         .         •"      •       "•'        I  part 

Absolute  alcohol       .         .         ...         .         .     100  parts 

Water       .         . 200  parts 

(A.  B.  AUBERT.) 

An  important  stain  for  nuclei,  bone,  and  connective  tissue. 

Wash  with  absolute  alcohol  after  staining. 

Victoria  blue.  —  Make  a  saturated  solution  of  this  stain  in 
water.  Wash  after  staining  in  alcohol  and  clear  in  oil  of  cloves 
(Lee).  This  is  a  nuclear  stain  and  gives  beautiful  results  and  is 
easy  to  manage.  Lee  says  that,  the  stain  has  a  strong  affinity 
for  elastic  tissue,  and  for  this  use  it  must  be  dissolved  in  alcohol 
diluted'  with  two  or  four  parts  of  water. 

In  the  process  of  coloring  objects  it  is  necessary,  in  order  to 
get  the  best  -results,  that  the  tissue  should  be  dead,  because 
living  tissue  will  not,  as  a  general  rule,  take  up  the  stains  so 


STAINS,  THEIR   PREPARATION  AND   USE 


109 


readily,  for  the  cells  seem  to  repel  the  coloring  matter.  It  has 
been  found,  therefore,  advisable  first  to  fix,  or  artificially  kill, 
the  specimen  before  the  sections  have  been  cut ;  this  is  particu- 
larly true  in  reference  to  animal  subjects,  and  in  the  case  of 
portions  of  plants  where  the  centres  of  life  will  totally  change 
unless  they  are  quickly  fixed  in  the  form  naturally  assumed 
during  the  living  period. 

For  a  detailed  account  of  the  processes  for  killing  and  hard- 
ening tissues,  see  page  71. 

How  to  stain.  —  Determine  first   what   portion   of  the  object 
you  wish  to  bring  out  for  study,  and  then  select  the  character  of 
stain  which  has  an  affinity  for  that  tissue, 
and  then  proceed  as  follows  :  — 

Dissolve  some  of  the  dry  stain  in  alcohol, 
or  water,  depending  on  the  condition  of  the 
object  and  the  character  of  the  stain,  and 
place  the  solution 
in  a  Naples  stain- 
ing-jar  (Fig.  61),  if 
the  sections  are 
fixed  to  the  slips ; 
if,  however,  the  sec- 
tions are  fixed  to  the 
cover-glasses,  it  will  be  best  to  use  a  dish  something  like  Moore's 
staining-dish  (Fig.  62),  which  has  parallel  ridges  for  holding  the 
cover-glasses,  and  a  cover-dish  for  keeping  out  dust.  This 
dish  is  so  constructed  that  it  may  be  used  for  holding  slides 
also. 

Now  having  fixed  the  section  securely  to  the  slip  or  the 
cover-glass,  by  one  of  the  methods  described  above,  and  having 
dissolved  the  paraffin  used  in  imbedding,  place  the  section  and 
its  slip  in  alcohol  for  a  short  time,  of  strength  of  95  per  cent, 
and  then  transfer  to  the  staining-dish,  if  the  stain  was  dissolved 
in  alcohol,  and  permit  the  slip  to  remain  in  this  solution  until 
the  section  is  fully  colored  to  the  intensity  desired.  The 
length  of  time  will  depend  on  the  character  of  the  section  and 


FIG.  61.  —  Naples  Stain- 
ing-jar. 


FIG.  62.  — Moore's  Staining- 
dish. 


HO  BIOLOGICAL  LABORATORY   METHODS 

the  stain  used.  These  points  can  be  determined  by  reference 
to  the  detailed  account  given  of  the  stain  elsewhere  in  this  book. 
If  the  stain  was  dissolved  in  water,  it  will  be  necessary  to  trans- 
fer the  section  and  the  slip  from  the  95  per  cent  alcohol  to  a 
vessel  containing  water  and  allowed  to  remain  for  a  few  minutes 
and  then  placed  in  the  vessel  containing  the  stain.  The  general 
rule  fcTr  transferring  from  alcohol  to  water  is  to  pass  the  section 
first  through  75  and  50  per  cent  alcohol,  and  then  to  water,  to 
prevent  injury  to  the  delicate  cells  of  the  specimen ;  but  some 
American  authorities  claim  that  this  is  not  necessary  if  a  very 
large  amount  of  water  is  used,  for  the  alcohol  is  taken  out  so 
quickly,  there  is  no  time  for  the  alcohol  and  water  to  mix  in  the 
section,  and  thus  diffusion  currents  are  avoided. 

After  the  stain  has  penetrated  long  enough  to  give  the  desired 
intensity,  the  extra  color  is  washed  off  the  section  with  alcohol 
or  water  (depending,  of  course,  upon  which  solution  of  the  stain 
is  used),  for  two  or  three  minutes,  and  then  dehydrated  in  95  per 
cent  alcohol.  The  dehydrating  will  take  from  a  few  minutes  to 
several  hours.  (See  Dehydrating.) 

Double  or  compound  staining.  —  Beautiful  results  are  secured 
by  this  process.  The  object  of  double  staining  is  so  to  differen- 
tiate the  tissues  as  to  bring  out  in  striking  contrast  each  group 
of  fibre  in  the  section  and  thus  facilitate  the  study  of  them  under 
the  microscope.  Double  staining  is  accomplished  by  the  use  of 
two  dyes,  one  to  stain  the  nucleus  and  the  other  to  color  the 
protoplasm  around  the  nucleus  or  the  cell  walls  surrounding. 
The  method  is  the  same  as  that  used  in  simple  staining,  and  the 
following  represent  some  of  the  most  important  combinations 
for  this  purpose  :  — 

1.  Carmine-anilin  blue.     Clear,  with  turpentine  and  mount 
in  balsam.     Suitable  for  all  kinds  of  tissue  and  nerve  centres. 
When  staining  in  mass  use  in  dilute  condition. 

2.  Hgematoxylin-eosin.     The  sections   should  be  very  thor- 
oughly washed  after  staining  in  the  eosin  and  before  transferring 
to  the  haematoxylin,  since  the  presence  of  the  former  will  pre- 
cipitate the  latter.     Stains  embryological  sections. 


STAINS,  THEIR  PREPARATION  AND   USE  III 

3.  Safranin-haematoxylin.      This  combination  gives  beautiful 
results.     Stain  first  with  haematoxylin,  very  slightly,  then  wash 
out  with  water  followed  by  alcohol  acidulated  with  hydrochloric 
acid,  and  stain  for  some  hours  with  safranin.     Stains   nuclei 
cytoplasm  beautifully. 

4.  Safranin-gentian. 

5.  Gentian-violet  eosin. 

6.  Methyl-violet  eosin. 

7.  Eosin  and  methyl-green. 

8.  Bismarck  brown  and  methyl-green. 

9.  Dahlia  and  eosin. 

Resegotti  has  found  by  experiment  that  those  stains  which  do 
not  select  the  nucleus  will  wash  out  from  the  fibres  the  stains 
which  are  purely  nuclear  colors.  He  has  classified  them,  there- 
fore, into  the  following  groups  :  — 

Nuclear  stains.  —  Safranin,  dahlia,  methyl- violet,  gentian  vio- 
let, magenta  and  Victoria  blue.  These  are  washed  out  by  the 
stains  mentioned  in  the  next  paragraph. 

The  secondary  or  non-nuclear  stains  are  methyl-green,  iodine 
green,  Congo,  orange,  acid  fuchsin,  cyanin,  eosin,  methyl-eosin, 
bordeaux,  and  vesuvin. 

It  will  be  of  importance  to  note  that  the  properties  possessed 
by  the  last  group  to  wash  out  those  found  in  the  first  list  will 
enable  the  student  to  procure  beautiful  contrasts  by  the  chemi- 
cal decoloration  one  of  these  colors  produces  on  the  other.  It 
must  be  borne  in  mind,  however,  that  the  washing-out  process 
must  not  be  prolonged,  otherwise  the  color  in  the  nucleus  will 
be  attacked  and  the  double  staining  will  be  destroyed.  Trans- 
ferring the  sections  to  water  will  stop  the  elimination  of  the 
color ;  or  they  may  be  cleared  and  mounted  at  once.  Clove  oil 
may  be  used  for  the  clearing  agent,  but  it  will  also  extract  some 
more  of  the  color,  and  this  fact  must  be  remembered  or  the 
section  will  present  a  faint  contrast  when  mounted.  Cedar  oil, 
xylol,  and  bergamot  oil  do  not  attack  the  stain,  and  they  may  be 
used  instead  of  the  clove  oil. 

In   the  Journal  of  the  Royal  Microscopical  Society  for   Octo- 


112  BIOLOGICAL  LABORATORY   METHODS 

her,  1893,  the  following  process  for  double-staining  vegetable 
tissue  is  given  :  — 

The  sections  are  first  decolored  by  eau-de-Javelle  (hypo- 
chlorite  of  potash),  and  then  left  for  a  quarter  of  an  hour  in  a 
concentrated  alcoholic  solution  of  cyanin,  then  washed  with 
absolute  alcohol  and  placed  for  a  quarter  of  an  hour  in  a  5  per 
cent  ammoniacal  solution  of  Congo  red.  After  washing  in  alcohol 
and  mounting  in  xylol  Canada  balsam,  the  sections  present  a 
magnificent  double-staining;  the  liquefied  membranes  are  col- 
ored an  intense  blue,  and  the  cellulose  membranes  are  dyed  rose 
color  or  red. 

To  stain  in  bulk  the  following  stains  have  been  found  suitable : 
carmine  (borax  and  alum),  cochineal,  haematoxylin,  Mayer's 
para-carmine,  Grenacher's  borax-carmine  (concentrated  solution 
of  carmine,  2  to  3  per  cent  borax  diluted  with  equal  bulk  of 
alcohol  of  70  per  cent),  Mayer's  carmalum. 

Haematoxylin  is  one  of  the  best  stains  for  staining  in  bulk, 
particularly  with  those  tissues  which  have  been  treated  with 
osmic  and  chromic  solutions.  It  is,  however,  very  difficult  to 
make  solutions  of  this  stain  which  will  keep  for  any  great 
length  of  time.  This  makes  the  staining  properties  of  hsema- 
toxylin  very  uncertain  unless  fresh  solutions  are  used  each 
time.  In  accordance  with  the  experiments  conducted  by  Mayer, 
he  has  found  that  it  is  best  to  use  haematein  instead  of  haematoxylin, 
which  avoids  the  tedious  method  formerly  used  in  "  ripening  " 
the  haematoxylin,  because  the  haematein  is  already  in  a  ripened 
condition.  Because  of  the  difficulty  at  times  in  securing  the 
haematein  in  a  sufficiently  pure  form,  Lee  recommends  the  use 
of  haematein-ammonia.  This  latter  stain  can  be  purchased  from 
dealers  in  dyes.  The  preparation  of  Mayer's  solution  is  as 
follows :  — 

MAYER'S  H^MALUM  :  — 

Hsematein  or  hsematein-ammonia         ...  I  gramme 

Alcohol  90  per  cent 50  cc. 

Dissolve  with  heat  and  add  to  solution  of  50  grammes  of  alum  in  a  litre  of 
water.  Filter  and  add  a  crystal  of  thymol  to  preserve  the  stain. 


STAINS,   THEIR  PREPARATION  AND   USE  1 1 3 

The  action  of  this  stain  is  quick,  and  specimens  which  are 
placed  in  it  when  it  is  in  a  concentrated  condition  are  stained  in 
a  few  moments.  It  acts  well  either  in  the  concentrated  or 
diluted  form.  It  is  highly  indorsed  for  staining  in  bulk.  Wash 
out  with  water  after  the  staining  is  completed.  In  clearing  do 
not  use  oil  of  cloves,  but  clear  with  oil  of  bergamot,  and  then 
remove  this  oil  with  oil  of  turpentine. 

DELAFIELD'S  H^EMATOXYLIN  STAIN:  — 

Hsematoxylin  crystals 4  grammes 

Absolute  alcohol 25  cc. 

Ammonium  alum        .         .         .         .         .         .52  grammes 

Distilled  water 400  cc. 

Glycerin     .        .         .         .        .         .         .         .  100  cc. 

Methyl  alcohol 100  cc. 

Dissolve  the  haematoxylin  in  the  absolute  alcohol,  the  alum 
in  hot  water,  and  then  add  the  haematoxylin  solution  drop  by 
drop  to  the  alum  solution  after  it  cools.  Expose  to  the  action 
of  the  light  and  air  for  three  or  four  days  until  the  liquid  turns 
dark,  filter  and  add  the  glycerin  and  methyl  alcohol.  Keep  in 
a  tightly  stoppered  bottle.  For  staining,  dilute  with  five  to  ten 
volumes  of  water. 

It  is  better  to  allow  this  stain  to  ripen  for  a  month  or  more 
before  using.  It  will  keep  for  years  and  is  a  very  strong  stain. 
It  is  excellent  for  embryos  and  tissues  en  masse.  It  forms  a 
good  combination  with  safranin,  the  haematoxylin  staining  the 
cellulose  a  rich  purple,  while  the  safranin  gives  to  the  lignified 
tissues  a  red  color. 

This  solution  is  also  a  stain  for  chromatin,  and  it  is  sometimes 
used  for  staining  nuclei. 

EHRLICH'S  H^MATOXYLIN  STAIN:  — 

Hsematoxylin      .......  2  grammes 

Absolute  alcohol 100  cc. 

Distilled  water 100  cc. 

Acetic  acid  (glacial) 10  cc. 

Glycerin 100  cc. 

Alum  in  excess 
I 


114  BIOLOGICAL   LABORATORY   METHODS 

Ripen  in  the  air  until  the  solution  turns  dark.  Keep  in  well- 
stoppered  bottle.  It  retains  its  properties  for  years.  It  stains 
in  bulk,  and  there  is  no  danger  of  overstaining.  It  acts  with 
great  rapidity  on  sections,  staining  them  in  a  few  minutes  a  deep 
color.  This  stain  has  great  affinity  for  chromatin. 

Heidenhain's  iron  haematoxylin  stain.  —  There  are  two  solutions 
comprising  this  stain,  and  they  must  be  kept  separate.  The  first 
consists  of  a  1.5  per  cent  solution  of  ferric  alum  or  ammonia 
sulphate  of  iron.  Care  must  be  exercised  to  use  the  double  salt 
of  the  sesquioxide  of  iron  and  not  the  protoxide.  The  crystals 
must  have  a  clear  violet-color.  The  second  solution  is  made  by 
dissolving  haematoxylin  in  water,  a  0.5  per  cent  solution. 

To  stain,  first  treat  the  specimens  in  the  ferric-alum  liquid  for 
a  half-hour  to  two  or  three  hours.  Wash  with  water.  The 
ferric-alum  is  not  the  stain  but  "is  simply  used  as  a  mordant  to 
prepare  the  specimens  for  the  second  solution,  which  is  the 
stain.  After  washing,  place  the  tissue  in  the  second  solution  for 
a  half -hour  and  wash,  and  again  treat  with  the  mordant,  which 
washes  out  the  stain  slowly,  until  the  desired  differentiation  is 
obtained.  The  changes  must  be  watched  under  the  microscope. 
A  short  immersion  in  the  haematoxylin  will  produce  blue  prepa- 
rations, while  a  prolongation  of  the  staining  will  give  black 
colorations.  The  blue  color  brings  out  strongly  the  nucleus, 
and  the  cytoplasmic  tissues  are  shown  by  lighter  shades.  The 
black  color  obtained  by  long  staining  shows  clearly  the  chromo- 
somes, the  central  corpuscles  staining  deep  black,  red  corpuscles 
black,  while  the  cytoplasm  will  generally  remain  colorless,  or 
will  turn  gray. 

This  stain  is  to  be  used  only  with  sections  which  must  be  very 
thin.  After  staining  wash  with  water,  then  pass  through  several 
changes  of  alcohol  of  varying  strengths,  clear  in  xylol,  and  mount 
in  balsam. 

Weigert's  method  for  staining  the  central  nervous  system  with 
haematoxylin.1  —  The  specimen  to  be  stained  must  first  be  hard- 

1  Dr.  B.  Pollack  in  Arch.f.  Mikr.  Anat.  (also  see  Jour.  Roy.  Mic.  Soc.,  p.  837, 
1897)"  calls  attention  to  the  importance  of  Weigert's  painstaking  work  on  the  human 


STAINS,   THEIR   PREPARATION  AND   USE  115 

ened  in  Miiller's  fluid,  or  Erlicki's  solution,  or  in  a  preparation 
of  formaldehyde.  This  hardening  process  takes  from  twenty-four 
hours  to  ten  days,  depending  upon  which  method  is  adopted  for 
the  purpose.  (See  chapter  on  hardening  solutions  and  methods.) 
The  tissue  is  then  transferred  to  grades  of  alcohol  without  wash- 
ing. Imbed  in  celloidin,  which  hardens  in  80  per  cent  alcohol. 
The  celloidin  block  is  then  mordanted  in  a  water  solution  con- 
taining neutral  acetate  of  copper,  5  per  cent ;  acetic  acid,  5  per 
cent ;  and  2\  per  cent  chrome  alum.  Make  this  solution  by  add- 
ing the  chrome  alum  to  hot  water  and  then  in  succession  the 
acetate  of  copper  and  the  acetic  acid.  The  mordanting  must 
continue  for  four  or  five  days.  Wash  in  alcohol,  cut  the  sections, 
and  then  stain  in  the  following :  — 


I.    Hsematoxylin  crystals 
Absolute  alcohol    . 

I  gramme 
10  cc. 

Water    . 

100  CC. 

For  use  mix  just  before  needed.  Continue  straining  action 
for  two  to  twenty-four  hours,  the  duration  depending  on  the 
character  of  the  tissue.  After  the  stain  has  acted  long  enough, 
wash  off  the  excess  of  the  solution  and  transfer  the  tissue  to  the 
following  decolorizer :  — 

Potassium  ferricyanide  .         .         .         .         .         2.5  grammes 

Borax    .         .         .         .         .         .         .         .         2     grammes 

Water .         .     100     cc. 

When  this  decolorizer  has  acted  long  enough,  which  is  indi- 
cated by  the  blue-black  condition  of  the  medullated  nerve -fibres 

neuroglia.  Golgi's  method  is  applicable  to  animals  almost  exclusively,  and  espe- 
cially to  embryos  ;  Weigert's  method  is  applicable  to  man.  Golgi's  method  colors 
only  some  cells  ;  Weigert's  colors  all  nuclei  and  fibres.  Golgi's  method  yields  a 
silhouette ;  Weigert's  shows  the  normal  form  and  size.  Golgi's  method  distinguishes 
only  cells  with  processes  ;  Weigert's  method  shows  cell  body,  or  nucleus  differ- 
entiated from  the  processes.  Golgi's  method  colors  besides  the  neuroglia  the 
nervous  tissues  ;  Weigert's  method  differentiates  the  neuroglia  —  a  fact  which  in 
part  explains  how  Golgi,  Caljal,  and  most  anatomists  hold  the  neuroglia  to  be 
nervous,  which  Weigert,  like  Ranvier,  vigorously  denies." 


Il6  BIOLOGICAL   LABORATORY   METHODS 

and  the  yellow  tint  given  the  gray  matter,  the  tissue  must  be 
washed  for  some  time  in  running  water,  dehydrated,  cleared  in 
oil  of  cloves,  and  mounted  in  balsam. 

This  method  is  not  suitable  for  staining  in  toto  because  the 
haematoxylin  will  not  penetrate. 

Golgi's  bichromate  and  nitrate  of  silver  method  for  staining  the 
nervous  system  (rapid  method).  —  A  hardening  mixture  is  first 
used  in  which  the  nervous  tissue,  in  small  portions,  is  soaked  for 
about  two  days  in  the  dark.  The  specimens  should  be  small 
and  the  quantity  of  the  hardening  solution  liberal  in  volume. 
Care  must  be  taken  neither  to  over  nor  under  harden,  for  in  the 
former  case  there  will  be  no  impregnation  of  the  silver,  and  in  the 
latter  condition  the  silver  will  diffuse  too  much.  The  character 
of  the  animal  must  determine  the  length  of  time  in  which  the 
tissues  are  subjected  to  the  action  of  the  hardening  solution. 

HARDENING  FLUID  :  — 

Bichromate  of  potassium  of  2  to  2.5  per  cent  strength       8  parts 
Osmic  acid  of  I  per  cent  strength      .    f    .         .         .         2  parts 

After  hardening  as  above  indicated,  the  specimens  are  brought 
into  a  weak,  or  used,  solution  of  silver,  so  that  precipitation  will 
not  occur  when  the  full  strength  of  the  fluid  is  used.  Wash 
until  precipitates  cease,  and  then  pour  on  a  liberal  quantity  of 
the  following  impregnation  solution  : — 

Silver  nitrate  crystals      .         .         .        .         .         0.75  grammes 
Distilled  water        .         .         .         ...    IOO       cc. 

In  winter  perform  the  impregnation  in  a  warm  place,  and  per- 
mit the  specimens  to  soak  for  a  period  of  24  to  48  hours.  The 
length  of  time  is  not  so  important  after  the  first  24  hours  ;  speci- 
mens may  remain  in  the  liquid  for  several  days  without  harm, 
provided  the  solution  is  renewed. 

The  specimens  are  now  washed  in  95  per  cent  alcohol,  and 
the  washing  is  repeated  until  all  of  the  free  nitrate  of  silver  is 
washed  out.  The  alcohol  must  remain  transparent  after  the 
specimens  have  been  in  it  for  two  or  three  days.  The  sections 


STAINS,  THEIR   PREPARATION  AND  USE  117 

are  now  cut  by  the  celloidin  method.  Place  the  celloidin  blocks 
in  absolute  alcohol,  changing  several  times,  clear  with  creosote 
for  a  few  minutes,  then  in  oil  of  bergamot  or  oil  of  turpentine 
for  fifteen  minutes.  Or  the  clearing  mixture  may  consist  of  car- 
bolic acid,  oil  of  bergamot,  and  oil  of  cedar  wood,  equal  parts,  in 
which  the  clearing  is  accomplished  rapidly.  Cut  the  sections 
and  mount  in  damar  without  the  cover-glass,  for  it  has  been 
found  that  those  sections  which  are  mounted  under  cover- 
glasses  lose  their  properties  and  soon  ruin.  The  drying  of  the 
damar  should  be  hastened  by  means  of  heat. 

Golgi's  method  stains  axis-cylinders,  ganglion  cells,  and  neu- 
roglia  cells  in  the  order  given.  In  the  use  of  the  stain  sometimes 
troublesome  precipitates  form  at  the  surface,  which  are  injurious 
to  the  clearness  of  the  image.  To  avoid  this  Sehrwald  used 
gelatin  in  the  following  manner :  The  specimen  was  placed  in  a 
paper  box  containing  a  10  per  cent  solution  of  gelatin  and  water 
before  impregnating  with  silver.  A  gentle  heat  was  applied  to 
the  gelatin  until  it  was  melted  and  the  specimen  became  well 
immersed.  The  mass  was  then  brought  into  the  impregnating 
solution,  and  after  the  silvering  the  gelatin  was  removed  by  means 
of  warm  water  saturated  with  chromate  of  silver. 

According  to  Dr.  G.  C.  Huber1  permanent  preparations  of 
nervous  tissue  stained  by  Golgi's  method  may  be  obtained  in  the 
following  manner :  — 

The  pieces  of  the  nervous  tissue  are  to  be  hardened  and  silvered 
according  to  the  procedure  advised  by  Ramon  y  Cajal2  and 
von  Kolliker,  and  celloidin  sections  cut  under  95  per  cent  alcohol. 
The  sections  are  then  immersed  for  fifteen  minutes  in  creosote 
and  then  for  some  minutes  in  turpentine.  They  are  now  spread 
out  on  a  slide,  pressed  down  with  filter-paper,  and  covered  with 
turpentine  balsam.  The  slide  is  then  gradually  and  carefully 
heated  over  a  flame  until  the  balsam  will  become  hard  when 
cooled.  At  this  stage  the  cover-glass  is  put  on  the  hot  balsam. 
The  heating  takes  from  three  to  five  minutes. 

1  Anat.  Anzeig.,  VII,  p.  587,  1892;  Jour.  Roy.  Mic.  Soc.,  p.  707,  1892, 

2  Lee's  "  Vade-Mecum,"  5th  ed.,  p.  418. 


Il8  BIOLOGICAL  LABORATORY   METHODS 

Dr.  Kopsch1  suggests  a  modification  of  Golgi's  method 
as  follows :  The  fixative  is  a  mixture  of  40  cc.  of  a  3.5  per 
cent  solution  of  potassium  bichromate  and  10  cc.  of  formalin. 
The  specimens  are  placed  in  this  fluid  for  twenty-four  hours,  and 
then  in  a  pure  solution  of  bichromate,  where  they  are  allowed  to 
remain  two  or  three  days.  The  specimens  are  then  transferred 
to  0.75  per  cent  silver  nitrate  solution  for  three  to  six  days. 
Nervous  tissue  thus  stained  does  not  suffer  from  precipitates. 

When  carmine  is  used  for  staining  en  masse,  the  washing  out 
must  be  performed  with  a  weak  solution  of  hydrochloric  acid 
(about  o.i  per  cent). 

Mayer's  carmalum,  or  alum-carmine,  is  prepared  as  follows  :  — 

Carminic  acid     .         ..        .         .         ...         I  gramme 

Alum        ,.         .         .         ,.        .         .         .         .       10  grammes 

Distilled  water  .        .        .        .        v        .    '     .     200  cc. 

Dissolve  with  heat  and  filter. 

Add  a  few  crystals  of  thymol  to  preserve. 

Wash  out  with  water.  Before  staining  in  bulk  see  that  the 
specimens  are  not  alkaline,  because  if  so  the  stain  will  not  act 
well.  This  is  an  excellent  stain. 

Gram's  method  for  staining  bacteria.  —  By  this  method  the  bac- 
teria are  stained  while  the  enclosing  tissues  remain  unstained. 
The  bacteria  are  stained  a  dark  blue,  and  they  are  brought  out 
very  clearly  from  the  surrounding  tissues. 

The  dyes  which  are  suitable  for  this  method  are  the  anilin 
colors,  especially  gentian  violet  and  fuchsin. 

The  original  plan  devised  by  Gram  was  first  to  stain  in  Ehr- 
lich's  anilin  water  gentian  violet  and  then  treat  with  iodine. 
The  anilin-water,  gentian-violet  solution  is  prepared  as  follows : 

Make  a  saturated  solution  of  anilin  oil  with  water  and  filter. 
Add  to  this  a  saturated  solution  of  gentian  violet  in  alcohol  until 
a  dark  color  is  obtained ;  it  will  take  about  i  cc.  of  the  gentian 
solution  to  20  cc.  of  the  anilin  water  to  accomplish  this.  Or 
we  may  prepare  the  stain  as  follows  :  — 

1  Anat.  Anzeig.,  XI,  p.  727,  1896. 


STAINS,  THEIR  PREPARATION  AND   USE       t 

Anilinoil          ......         •  3  parts 

Water       .........  80  parts 

Gentian  violet  ........  l  Part 

Alcohol    .........  *5 


The  specimens  of  bacteria  fixed  to  the  cover-glass  are  first 
placed  in  absolute  alcohol,  and  then  transferred  to  the  stain  for 
a  few  minutes,  where  they  become  deeply  stained.  The  cover- 
glass  is  now  placed  in  a  weak  solution  qf  iodine  and  iodide  of 
potassium,  prepared  as  follows  :  — 

Iodine        ........         I  gramme 

Iodide  of  potassium  ......         2'  grammes 

Water         ..'•'.'     ......     300  grammes 

The  section  remains  in  this  solution  from  one  to  three  min- 
utes, and  is  then  placed  in  absolute  alcohol  until  nearly  all  of 
the  color  is  washed  out  ;  in  fact,  the  section  seems  to  have  become 
decolorized  entirely.  Clarify  in  clove  oil  and  mount.  Under 
the  microscope  the  bacteria  will  be  seen  colored  a  beautiful  dark 
blue  and  the  nuclei  without  stain.  The  nuclei,  however,  may  be 
also  stained  by  following  the  above  method  with  Bismarck  brown, 
thus  giving  the  nuclei  a  shade  of  yellow  or  brown,  with  the  bac- 
teria still  a  dark  blue  and  very  distinct. 

It  is  unfortunate  that  all  bacteria  will  not  stain  under  the 
Gram  method  with  the  same  degree  of  success,  but  so  many  of 
them  yield  to  this  treatment  that  it  has  become  one  of  the  stand- 
ard methods  of  staining  among  bacteriologists. 

"  Intra  vitam"  staining.  —  This  term  is  used  to  apply  to 
the  staining  of  living  tissue,  without  destroying  the  life  of 
the  organism.  The  method  is  of  great  advantage  in  deter- 
mining certain  physiological  conditions  that  would  be  im- 
possible of  satisfactory  solution  without  the  use  of  this  method. 
The  stain  must  be  considerably  diluted,  however,  and  feebly 
alkaline  or  neutral.  The  strength  of  the  stain  ranges  from 
i  :  10,000  to  i  :  100,000,  depending  upon  the  character  of 
the  animal  to  be  treated  and  the  tissue  to  be  stained.  It 
is  best  to  experiment  by  starting  with  a  strength  of  i  :  100,000, 
and  increasing  the  coloring  matter  until  the  desired  strength 


120      .  BIOLOGICAL  LABORATORY   METHODS 

is  secured.  If  the  animal  is  aquatic  in  habits,  the  staining  can 
be  accomplished  by  adding  methylene-blue  to  the  water  in  which 
the  animal  is  living.  Certain  of  the  tissues  are  colored  by  this 
method,  such  as  cytoplasm  of  some  cells,  sensory  nerves,  and 
lymph  spaces.  Marine  animals  are  also  satisfactorily  examined 
under  this  method  by  simply  adding  the  methylene-blue  to  the 
sea-water  in  which  the  animal  is  living,  and  during  its  life  the 
coloring  matter  is  taken  into  its  system.  It  is  important,  how- 
ever, in  this  class  of  work  to  note  just  when  the  special  tissue 
is  stained  its  maximum,  because  the  animal  begins  to  throw  off 
the  stain  after  a  certain  period  and  soon  leaves  the  tissues  in 
their  normal  condition. 

In  some  cases  it  is  necessary  to  inject  the  staining  solution 
into  the  living  animal  to  secure  the  desired  results.  The  injec- 
tion is  made  into  the  vascular  system  or  the  body  cavity  of  the 
living  animal,  and  as  soon  as  the  coloring  matter  has  had  time 
to  penetrate  the  organs  and  stain  the  tissues  under  study,  these 
tissues  are  extracted  and  mounted  for  preservation. 

The  following  stains  have  been  found  to  yield  good  results  by 
the  infra  vitam  method :  Methylene-blue,  Congo  red,  Bismarck 
brown,  cyanin,  dahlia,  gentian  neutral  red,  methyl-violet,  safranin, 
and  malachite-green.  Methylene-blue  gives  the  best  results  on 
the  sensory  nerves  ;  for  Infusoria  use  Bismarck  brown  ;  and  for 
cell  tissues  use  Congo  red,  methylene-blue,  gentian  violet,  and 
dahlia.  The  methylene-blue  stain  is  specially  valuable  in  the 
investigations  of  the  tannin  vesicles;  the  vesicles  are  separated 
from  the  other  tissue  with  a  distinct  deep  blue  color. 

To  fix  the  stain  for  preservation  transfer  to  a  concentrated 
aqueous  solution  of  picrate  of  ammonia  for  one-half  hour  to 
twenty-four  hours,  and  mount  in  pure  glycerin  (Dogiel).  In 
order  to  prevent  the  macerating  effects  produced  by  the  picrate 
of  ammonia  on  some  tissue,  there  is  added  to  the  fixing-bath  a 
i  per  cent  osmic  acid  solution  at  the  rate  of  i  to  2  per  cent  of  the 
bath.  Feist  has  claimed  that  if  picro-carmine  be  used,  the  blue 
color  is  well  preserved,  provided  the  action  of  the  picro-carmine 
is  not  allowed  to  continue  for  a  long  period. 


STAINS,  THEIR   PREPARATION  AND   USE  121 

Staining  infra  vitam  may  be  conducted  with  much  interest  to 
the  investigator  by  placing  the  living  forms,  if  minute,  on  a  glass 
slip  and  covering  with  a  suitable  size  cover-glass  resting  on  bits  of 
wax.  Permit  the  diluted  color  to  flow  gently  through  the  space 
between  the  glasses,  and  watch  the  results  through  the  micro- 
scope. When  the  desired  tint  is  obtained  in  the  tissues  of  the 
animal,  discontinue  the  flow  of  the  coloring  fluid  and  perma- 
nently fix  and  mount. 

It  has  been  discovered  by  Herr  A.  M.  Przesmycki 1  that  dif- 
ferent organisms  act  differently  in  intra  vitam  staining ;  some 
organs  even  in  the  same  animal  exhibit  less  receptivity  than  is 
shown  by  others.  He  also  proves  that  the  parts  colored  intra 
vitam  lose  their  color  after  death,  and  when  the  organism  is 
transferred  to  fresh  water  the  stain  is  washed  out. 

Paul  Mayer 2  likewise  proves  that  Bismarck  brown  dissolved 
in  sea-water  and  Caprellidae  placed  in  the  solution,  the  contents 
of  the  stomach  and  the  hepatopancreas  were  stained  brown. 
Transferred  to  pure  sea-water  the  specimens  lost  their  coloring 
in  a  few  hours,  and  the  animals  seemed  to  be  unharmed. 

Professor  P.  Ehrlich, 3  one  of  the  earlier  investigators  on  intra 
vitam  staining,  gives  the  following  items  concerning  vital  infu- 
sion of  nerves  with  methylene-blue  :  "  There  are  two  conditions 
necessary  to  get  the  methylene-blue  reaction  : 

"i.    Saturation  with  oxygen. 

"  2.    Alkaline  reaction. 

"  The  first  is  obtained  by  free  exposure  to  the  air.  To  secure 
the  second  condition  it  is  necessary  to  experiment  on  nerves  at 
rest,  since  it  is  well  known  that  when  in  that  condition  they  are 
alkaline  in  reaction.  This  condition  may  be  obtained  by  sever- 
ing the  nerves  before  applying  the  methylene-blue  or  by  poison- 
ing the  animal  with  curare.  The  method  of  procedure  is  as 
follows :  — 

"  Inject  the  vena  cutanae  magna  of  a  frog  with  i  cc.  of  a 

1  Biol.  Cent.,  XVII,  p.  353,  1897. 

2  Fauna  u.  Flora  des  Golfes  von  Neapel  vi  Monographic,  p.  153,  1882. 

3  Deut.  Med.  Wochenschr.,  No.  4,  1886. 


122  BIOLOGICAL  LABORATORY  METHODS 

saturated  solution  of  methylene-blue.  The  tongue  and  palate  are 
at  once  colored,  but  the  stain  is  confined  to  the  blood-vessels, 
and  does  not  at  first  affect  the  nerves.  After  an  hour  or  two  the 
nerves  supplying  the  taste  papillae  appear  blue,  and  meshes  of 
the  palate  are  also  stained.  The  motor  nerve-ends  show  the 
stain  a  little  later.  The  color  reaction  lasts  only  a  short  time, 
often  not  more  than  five  or  ten  minutes.  It  should  be  fixed  at 
the  moment  of  greatest  intensity.  If  iodine  is  used  for  this  pur- 
pose, proceed  as  follows:  — 

"  Place  the  frog  in  a  i  per  cent  aqueous  solution  of  potassic 
iodide,  in  which  metallic  iodine  has  been  dissolved  to  saturation, 
and  inject  the  blood-vessels  with  the  same  solution,  thus  freeing 
them  from  blood  as  far  as  possible.  Next  cut  out  the  parts  needed 
and  leave  them  in  the  iodine  solution  from  five  to  twelve  hours. 
Transfer  to  water,  and  leave  until  most  of  the  iodine  has  been 
withdrawn  from  the  specimen.  As  a  result  of  this  treatment  the 
nerves  will  have  a  dark  brown  or  gray  color,  and  the  surrounding 
tissue  will  be  nearly  colorless.  Mount  in  acidified  glycerin." 

Examples  of  how  to  stain  tissues.  —  The  following  list  of  tis- 
sues is  given  for  the  purpose  of  furnishing  the  student  some 
examples  of  how  the  stains  are  used  in  order  to  produce  the  best 
results  in  bringing  out  the  details  of  the  section  of  the  specimen 
under  investigation.  No  attempt  has  been  made  to  give  a  com- 
plete list  of  the  tissues,  but  a  sufficient  number  is  submitted  to 
enable  the  student  greatly  to  extend  the  list  after  he  has  famil- 
iarized himself  with  the  stains  mentioned  under  each  item. 

Adenoid  tissue.  —  Delafield's  haematoxylin  method. 

Albumen  crystals  in  plants.  —  Haematoxylin  produces  beauti- 
ful violet  colors. 

Aleurone  or  protein  grains.  —  Stain  with  iodine.  The  treat- 
ment with  chloral  hydrate  in  2  parts  of  water  in  which  a  little 
iodine  is  placed  discolors  the  chlorophyl  bodies,  and  causes 
them  to  swell  with  the  starch  grains,  and  the  starch  comes  out 
clearly  stained  blue.  Or  the  chlorophyl  bodies  may  be  dis- 
colored with  alcohol,  and  then  stained  with  dilute  aqueous  solu- 
tion of  methyl-violet. 


STAINS,  THEIR  PREPARATION  AND  USE  123 

Areolar,  adipose,  and  retiform  tissue.  —  Fuchsin  solution  mixed 
with  a  little  haemalum  gives  these  tissues  a  deep  stain. 

Articular  cartilage  and  synovial  membranes.  —  Treat  with  dilute 
haemalum  and  carmalum,  and  mount  in  dilute  glycerin. 

Axis  cylinders  and  protoplasm  nerves.  —  Either  of  the  following 
staining  methods  :  Golgi's  sublimate,  Golgi's  bichromate  of  silver 
method,  methylene-blue  method,  Weigert's  specific  neuroglia  stain. 

Bacteria.  —  Bismarck  brown,  safranin,  fuchsin,  gentian  violet, 
methylene-blue.  Spores  may  be  stained  by  Moeller's  method  as 
follows  :  First  place  in  absolute  alcohol  for  two  minutes,  then  for 
a  minute  in  a  5  per  cent  solution  of  chromic  acid ;  wash  and 
stain  in  Ziehl's  solution  for  one  minute  (5  per  cent  solution  in 
water  of  carbolic  acid,  to  which  is  added  about  one-tenth  its 
volume  of  saturated  solution  in  alcohol  of  fuchsin).  The  stain 
is  heated  to  boiling.  Place  the  spores  in  5  per  cent  solution  of 
sulphuric  acid  to  decolorize.  This  will  leave  the  spores  red  and 
the  germs  uncolored.  The  chromic  acid  acts  on  the  membranes 
of  the  specimen,  and  thus  permits  the  stain  to  penetrate  to  the 
spores.  After  fixation  the  fat  cholesterin  crystals  and  other  like 
materials  may  be  separated  from  the  spores  by  immersion  in 
chloroform  for  several  minutes.  Buchner  and  Hueppe  have  dis- 
covered (1884)  that  spores  do  not  take  the  stain  so  well  unless 
they  are  heated.  This  can  be  accomplished  by  placing  the  glass 
slip  or  cover-glass  on  which  the  spores  are  fixed  in  a  hot-air 
oven,  and  raising  the  temperature  to  120°  or  175°  C.  for  one  hour. 

Bacteria  in  tissue.  —  Weigert's  or  Gram's  method.  Weigert's 
method  is  to  dehydrate  in  anilin  oil,  wash  in  water,  place  on  the 
slide,  and  remove  the  excess  of  the  water  by  means  of  filter- 
paper,  and  then  pour  on  the  specimen  iodide  solution  (iodine,  i 
part ;  potassic  iodide,  2  parts  ;  water,  300  parts).  To  decolorize 
the  specimen,  remove  iodine  solution  with  filter-paper  or  blotter; 
dehydrate  with  a  few  drops  of  anilin  oil ;  remove  with  filter- 
paper;  then  treat  with  xylol  to  remove  all  traces  of  anilin  oil; 
then  mount  in  balsam.  The  basic  anilin  dyes  take  the  front  rank 
in  staining  bacteria. 

Blood.  —  Corpuscles  with   haemoglobin   stain    red   in  eosin ; 


in  st 

' 


124  BIOLOGICAL  LABORATORY   METHODS 

methyl-violet  stains  the  nuclei  of  the  corpuscles.  Ehrlich- 
Biondi's  method  is  excellent  for  this  purpose.  (To  100  cc. 
saturated  aqueous  solution  of  orange  add  with  agitation  20  cc. 
saturated  aqueous  solution  acid  fuchsin  and  50  cc.  of  a  like 
solution  of  methyl-green.  For  the  method  and  its  use,  see 
Lee's  "  Vade-Mecum,"  5th  ed.,  p.  215.) 

Bone.  —  Schaffer's  method:  The  bone  is  first  decalcified  in 
nitric  acid,  and  then  stained  for  one-half  hour  in  0.05  per  cent 
aqueous  solution  of  safranin,  washed  with  water,  and  then  treated 
for  two  or  three  hours  in  o.oi  per  cent  solution  of  corrosive  subli- 
mate, and  then  mounted  in  glycerin.  To  make  the  preparation 
permanent,  wash  in  alcohol  after  removal  from  the  sublimate, 
and  clear  for  a  long  time  in  bergamot  oil,  and  then  mount  in 
xylol  balsam. 

Bone  and  marrow.  —  One  per  cent  of  eosin  in  alcohol ;  dilute 
fuchsin  after  the  bone  has  been  decalcified  in  nitric  acid  or  in 
chromic  acid ;  mount  in  glycerin. 

Callus  in  sieve-plates.  —  Anilin  blue  or  fuchsin. 

Cell  contents.  — Haematoxylin. 

Cell  granules.  —  Ehrlich's  mixture. 

Cell  walls.  —  Methylene-blue  ;  alum-carmine  ;  eosin. 

Cellulose  tissue.  —  Aqueous  solution  of  anilin  green ;  the  cell 
walls  are  stained  green,  while  the  walls  of  parenchymatous  cells 
are  stained  bluish  green.  Chlorzinc  iodide  (Schultze's  solution  : 
Dissolve  pure  zinc  in  hydrochloric  acid,  evaporate  with  a  piece 
of  metallic  zinc  in  the  solution  to  a  strong  consistency,  add 
iodide  of  potassium  to  saturation,  and  finally  as  much  metallic 
iodine  as  the  solution  will  take  up)  added  to  a  vessel  of  water 
containing  a  section  of  the  stem  will  show  in  brownish  yellow 
color  the  cellulose,  while  the  pits  remain  unstained  and  thus 
stand  out  clear  and  distinct.  The  pits  are  traversed  by  violet 
striation,  and  here  and  there  on  them  are  granulations  stained 
yellow  brown.  The  cuticle  also  stains  a  yellowish  brown.  Grena- 
dier's alum-carmine  will  yield  good  results. 

Chlorophyl  grains.  —  Methyl-green. 

Chromatin  in  nucleus.  —  The  fixing  agents  for  the  nuclei  must 


STAINS,  THEIR   PREPARATION  AND   USE  125 

invariably  be  acid  in  order  to  preserve  the  chromatin.  Flem- 
ming's  and  Hermann's  liquids  are  best  for  this  purpose.  Methyl- 
green  is  a  good  stain  for  the  chromatin,  but  it  sometimes  acts 
very  weakly  in  intensity  in  different  nuclei.  Safranin  acts  well. 

Cork  cells.  —  Chloriodide  of  zinc  stains  yellow ;  fuchsin  dis- 
solved in  alcohol  is  also  good  stain. 

Cornea.  —  Methylene-blue  method. 

Cuticularized  layer  of  epidermis.  —  Chloriodide  of  zinc  colors 
this  tissue  yellow.  The  true  cuticle  is  uncolored. 

Echinoderms.  —  Methylene-blue  method  by  intra  vitam. 

Elastic  tissue.  —  Weigert's  stain  colors  dark  blue,  and  the 
other  tissues  remain  lighter  color. 

Fuchsin     .........  2  grammes 

Resorsin    .         .         .         .         .         .         .                   .'  4  grammes 

Liquor  ferri  sesquichlorati 30  cc. 

Water  distilled 200  cc. 

Embryos.  —  Delafield's  haematoxylin  is  excellent  for  staining 
small  embryos  of  mammalia.  For  staining  large  embryos  use 
carmalum,  and  also  Delafield's  acetic-acid-alum-carmine. 

Epithelium. —  Nitrate  of  silver ;  iron  haematoxylin  ;  methylene- 
blue. 

Fatty  oils.  —  Perosmic  acid  colors  them  black  or  brown. 

Ganglia.  —  Methylene-blue,  100  parts  to  i  part  of  salt  water, 
fix  in  picrate  of  ammonia,  and  mount  in  glycerin  or  balsam. 

Glands.  —  Liver  is  stained  in  Golgi's  rapid  method,  or  by 
Ehrlich-Biondi's  method.  The  kidney  glands  are  stained  with 
iron  haematoxylin,  and  afterward  in  a  solution  of  fuchsin  which 
brings  out  the  ciliary  plateau. 

Intercellular  bridges  and  canals.  —  Use  iron  haematoxylin. 

Lignin.  —  Anilin  chloride  in  dilute  solution  with  addition  of 
hydrochloric  acid.  The  color  is 'pale  yellow.  Chloriodide  of 
zinc  stains  a  yellow  color. 

Lymph  glands  and  cells.  —  Haematoxylin  and  eosin. 

Muscles.  —  Delafield's  haematoxylin.     Eosin. 

Nerve  fibres,  cells,  and  endings.  —  Methylene-blue  method ; 
Golgi's  bichromate  of  silver  method  ;  Weigert's  specific  neu- 
roglia  stain. 


126  BIOLOGICAL  LABORATORY  METHODS 

Nuclei  of  cells.  —  Carmine  in  dilute  potash  and  mixed  with 
alcohol ;  ammonia  carmine  is  also  generally  used,  prepared  by 
Hartig's  formula. 

Nucleus.  —  Ehrlich-Biondi's  stain  colors  red  or  orange,  and 
iron  haematoxylin  stains  black  or  gray;  Thiersch's  borax-car- 
mine ;  methyl-green ;  picro-carmine. 

Phloem  in  vibro-vascular  bundles.  —  Cochineal  with  acetic  acid 
colors  red. 

Plasma.  —  Equal  volumes  of  anilin  water  and  concentrated 
solution  of  methyl-violet  6  B.  Wash  and  treat  with  solution  of 
iodide  of  potassium,  wash  and  treat  with  mixture  of  i  volume  of 
anilin  to  2  volumes  of  xylol,  and  then  in  pure  xylol. 

Prosenchyma  bast  tissue.  —  Cochineal  with  acetic  acid. 

Protoplasm,  living.  —  Silver  nitrate,  in  dilute  solution,  colors 
the  protoplasm  dark.  Picro-carmine  gives  a  yellow-red  color. 

Protozoa. — Intra  vitam  method  with  methyl-green  acid  solution. 

Resin.  —  Miiller's  tincture  of  alcannin  gives  red,  and  methyl- 
violet  gives  blue  colors. 

Retina.  —  Pal's  modification  of  Weigert's  method. 

Salivary  glands.  —  Carmalum  or  Heidenhain's  haematoxylin 
method. 

Sieve-plates.  —  Anilin  blue,  place  in  glycerin,  and  the  latter 
will  extract  the  color  except  that  which  is  taken  up  by  the  sieve- 
plates,  and  they  come  out  in  a  beautiful  color. 

Skin.  —  Haemalum  or  carmalum.  Treat  with  picric  acid  and 
mount  in  xylol  balsam.  Borax-carmine  or  haematoxylin  or  eosin, 
and  clear  in  carbol-xylol. 

Spinal  cord.  —  Weigert's  haematoxylin  method,  and  cleared  in 
carbol-xylol. 

Spleen.  —  Haematoxylin  and  eosin,  or  methylene-blue. 

Sponges.  —  Mayer's  tincture  of  cochineal. 

Tannin.  —  Dilute  solution  of  chloriodide  of  zinc  colors  red  or 
violet. 

Vascular  bundles.  —  Chloriodide  of  zinc  colors  the  bast  a 
distinct  violet,  the  lignified  parenchyma  cells  surrounding  the 
bundle  are  stained  red-brown. 


CHAPTER  VII 

MOUNTING    THE    TISSUE    FOR    PRESERVATION 

Mounting  in  Canada  balsam.  —  After  the  sections  are  properly 
stained,  dehydrated,  and  fixed  to  the  slip,  it  becomes  necessary 
to  mount  them  in  some  medium  which  has  preservative  prop- 
erties ;  otherwise  decay  will  set  in,  and  in  time  the  sections  will 
be  ruined.  One  of  the  best  mounting  media  is  Canada  balsam 1 
dissolved  in  either  xylol,  benzole,  chloroform,  or  turpentine, 
because  of  its  refractive  properties  and  the  ease  with  which  it 
is  manipulated,  and  its  valuable  preserving  powers. 

In  the  use  of  this  medium,  it  is  absolutely  necessary  that  the 
specimens  be  thoroughly  dehydrated,  or  a  cloudiness  is  produced 
of  watery  vapor,  and  the  slide  will  be  sadly  ruined  until  the 
cover-glass  is  removed  and  a  new  mount  is  made. 

The  glass  generally  used  for  holding  the  object  is  made  of  the 
best  white  crown  glass  (Fig.  63),  measuring  76x26  mm.,  or 
48x38  mm.,  the  edges  of  which  are  ground.  The  cover-glasses 
are  thin  circles  (Fig.  64),  cut  to  dimensions  of  12,  15,  18,  21,  and 
24  mm.  Square  cover-glasses  (Fig.  65)  of  the  same  dimensions 
can  also  be  purchased  from  dealers,  when  such  shapes  are  de- 
sired. The  square  forms  are  not  suitable,  however,  when  the 
manipulator  wishes  to  use  the  turn-table  to  form  a  ring  before 
and  after  placing  on  the  cover-glass.  The  thickness  of  the 
cover-glasses  varies  from  0.15  to  0.22  mm.  When  the  immer- 
sion objective  is  used,  it  becomes  desirable  to  know  this  thick- 
ness. 

1  Datnar  is  also  used  for  mounting  sections  and  by  some  is  preferred  to  balsam ; 
Lee,  in  his  "  Vade-Mecum,"  states  that  "  damar  gives  the  better  definition  of  deli- 
cate detail;  balsam  has  greater  clearing  action,  and  affords  perhaps  more  solid 
mounts"  (p.  249). 

127 


MOUNTING  THE  TISSUE  FOR   PRESERVATION 


129 


Of  course  it  is  important  that  these  glass  slips  and  covers 
should  be  scrupulously  clean,  free  from  all  films  of  grease  and 
other  extraneous  matters,  before  the  sections  are  fixed ;  other- 
wise serious  trouble  awaits  the  operator.  The  cementing  ring 
will  fail  to  attach  firmly  to  the  surface,  and  the  mount  will  pre- 
sent a  slovenly  appearance.  For  recipes  suitable  for  cleaning 
the  glasses,  the  student  is  referred  to  the  pages  at  the  close  of 
this  volume. 

The  mode  of  procedure  for  making  the  complete  mount  is  as 
follows :  If  the  sections  do  not  require  a  previously  prepared 
ring  for  the  glass  cover,  place  the  slip  on  the  turn-table  and 
centre  accurately.  By  means  of  a  needle  put  a  drop  of  Canada 
balsam  on  the  sections,  and  the  proper  size  cover-glass  is  grasped 
between  the  forceps  and  the  edge  of  the  glass  cover  opposite  to 
the  forceps  is  first  brought  in  contact  with  the  slip  and  then 
gradually  lowered  until  nearly  in  contact  all  around,  when  the 
forceps  are  removed  and  the  circle  is  permitted  to  fall  gently 
into  place.  A  spring-clip  is  now  placed  on,  so  that  all  superflu- 
ous balsam  is  driven  out,  as  well  as  most,  if  not  all,  of  the  air 
bubbles.  If  a  few  very  small  air  bubbles  remain,  these  will  pass 
out  when  the  balsam  begins  to  thicken,  in  the  course  of  a  few 
hours. 

If  the  sections  are  too  thick,  however,  to  take  the  cover-glass 
without  a  ring  to  raise  the  cover  above  the  slip,  the  slip  should 
be  prepared  with  a  ring  some  time  before  the  sections  are  placed 
in  position,  in  order  to  give  the  gum  composing  it  time  to 
thoroughly  harden.  This  primary  ring  can  be  as  high  as  desired 
by  repeated  applications  after  each  coating  has  had  time  to  first 
dry  and  harden.  In  making  the  mount,  the  edges  of  the  cover- 
glass  should  reach  only  as  far  as  the  centre  of  the  ring,  and  it 
will  then  be  possible  to  seal  the  mount  with  the  last  ring. 

These  gummed  rings  are  made  of  either  Brunswick  black, 
shellac,  King's  cement,  asphalt,  or  other  like  solutions,  dissolved 
in  xylol,  benzole,  alcohol,  etc.  The  spinning  is  accomplished 
by  means  of  a  small  camel's-hair  brush,  held  with  a  steady 
hand  in  contact  with  the  glass  slip,  while  the  table  is  rapidly 


130  BIOLOGICAL   LABORATORY   METHODS 

whirled  by  the  other  hand.  The  brush  should  be  so  held  that  a 
line  drawn  from  the  person  through  the  centre  of  the  table  will 
make  nearly  a  right  angle  with  the  line  passing  through  the 
length  of  the  brush  handle.  The  cell  wall  should  be  one-sixth 
of  an  inch  wide,  and  should  project  about  half  that  space  beyond 
the  edge  of  the  cover-glass. 

After  seasoning  for  two  or  three  days,  or  longer  if  required, 
to  well  harden  the  balsam  between  the  cover-glass  and  the  slip, 
the  mount  is  placed  on  the  turn-table  again  and,  by  means  of  a 
scalpel,  the  balsam  remaining  outside  of  the  cover  is  carefully 
scraped  off  as  the  table  is  slowly  turned.  A  cloth,  moistened  in 
benzole  or  xylol,  is  rubbed  over  those  portions  of  the  glass  still 
containing  films  of  balsam,  extra  care  being  taken,  however,  to 
avoid  displacing  the  cover-glass  and  dissolving  the  hardened 
balsam  under  its  edge,  which  cements  it  to  the  glass  slip.  All 
balsam  must  be  removed  and  the  benzole  dried  off  to  prevent 
the  finishing  ring  from  running  when  it  is  applied.  When  this 
cleaning  is  thoroughly  completed,  the  cover-glass  must  be  per- 
manently sealed  with  a  ring  of  gum,  which  will  prevent  the 
access  of  air  or  displacement  of  the  cover  in  after  handling. 
This  finishing  ring  is  put  on  in  the  same  manner  described  for 
making  the  cell  wall;  and  the  precaution  is  taken  to  spin  with  a 
cement  which  has  a  solvent  different  to  that  in  which  the  balsam 
is  dissolved  to  prevent  its  running  under  the  edges  of  the  cover 
and  staining  the  balsam,  because  it  must  be  remembered  that 
the  same  solvent  will  dissolve  both.  For  instance,  benzole  and 
xylol  are  solvents  of  balsam ;  a  ring,  therefore,  made  of  a  gum 
dissolved  in  either  benzole  or  xylol  will  run  when  the  balsam 
comes  in  contact  with  it.  In  this  case,  then,  it  is  best  to  make 
the  ring  of  a  cement  which  is  soluble  in  alcohol,  like  King's,  or 
one  soluble  in  water.  In  spinning  the  final  ring,  bring  the  brush 
in  contact  with  the  circle  and  slip  at  the  same  time,  so  that  the 
ring  will  lap  on  each,  and  thus  make  a  perfect  sealing.  The 
slide  is  now  properly  labelled  and  placed  aside  to  dry.  If  the 
damar  cement  is  preferred,  the  steps  are  the  same  as  those  given 
in  the  case  of  balsam. 


MOUNTING  THE  TISSUE  FOR  PRESERVATION          131 

Glycerin  jelly  mounting.  —  The  fixing  of  the  sections  to  the 
slip  and  staining  are  in  all  respects  the  same  for  mounting  in 
glycerin  as  already  described  for  mounting  in  balsam.  A  small 
lump  of  the  glycerin  jelly  is  transferred  to  the  centre  of  the 
cover-glass,  and  warmed  in  the  water-bath.  As  soon  as  the 
glycerin  melts,  the  slip  also  warmed  is  centred  on  the  turn- 
table and  the  cover-glass  put  in  place  with  the  forceps  the  usual 
way  (see  page  129).  Care  must  be  taken  that  the  heating  in  the 
water-bath  is  not  greatly  prolonged,  because  there  wall  be  danger 
of  introducing  many  air  bubbles  in  the  glycerin.  If  air  bubbles 
should  appear,  they  must  be  exploded  before  the  cover-glass  is 
put  on,  and  a  close  examination  must  be  made  for  them  by 
means  of  the  hand  magnify  ing-glass.  By  placing  the  jelly  on 
the  cover-glass  instead  of  the  slip,  the  chances  for  air  bubbles 
are  greatly  reduced.  With  the  spring-clip  in  position  on  the 
slide,  the  surplus  glycerin  is  carefully  washed  off  with  water  and 
the  surface  of  the  glass  thoroughly  dried  by  means  of  blotters. 
A  ring  of  the  glycerin  jelly  is  now  spun  around  the  edges  of  the 
cover-glass  and  the  slide  is  put  aside  under  a  bell-glass  to  season 
and  harden,  after  which  the  finishing  gum  (dissolved  in  benzole 
or  xylol)  is  spun  over  the  jelly  and  the  slide  is  then  labelled. 
The  jelly,  in  each  case,  must  be  allowed  to  congeal  before  the 
gum  is  spun  on.  The  student  is  referred  to  the  chapter  on 
recipes  for  a  suitable  cement  for  finishing  his  slide. 

Dry  mounts.  —  It  is  only  necessary  to  use  mounting  media 
in  those  cases  where  preservation  is  demanded,  but  in  some 
instances  the  specimen  may  be  mounted  without  the  media, 
particularly  when  the  slide  is  intended  only  for  temporary  use, 
and  there  is  no  desire  permanently  to  preserve  the  form.  Under 
these  conditions  the  specimen  is  attached  to  the  slip  by  means 
of  some  transparent  cement,  —  Canada  balsam  will  do  very  well, 
—  and  a  thin  ring  of  cement  is  spun  around  the  well-seasoned 
cell,  and  the  cover-glass  is  firmly  pressed  in  contact  with  it,  and 
the  slide  is  then  placed  aside  to  dry,  after  which  the  finishing 
ring  is  applied  if  desired. 


CHAPTER  VIII 

HOW   TO    MAKE    DRAWINGS    OF   THE    SECTIONS    OF   TISSUE 

BEFORE  photography  became  so  widely  used  —  anterior  to  the 
introduction  of  dry  plates  and  the  many  facilities  for  rapid  and 
comparatively  easy  working  —  drawing  was  the  only  method 
available  for  readily  transferring  to  paper  what  was  revealed 
in  the  microscope.  Even  now  some  microscopists  of  distin- 
guished abilities  prefer  drawing  to  photographing  the  image. 
It  is  the  opinion  of  the  author  that  both  methods  should  be 
used  ;  drawing  in  some  cases  will  be  found  best,  while  in  others 
photography  will  give  the  most  satisfaction.  Drawing  must  be 
entirely  free-hand,  and,  of  course,  where  the  results  are  largely 
due  to  the  judgment  of  the  observer,  the  reproduction  cannot  in 
all  particulars  be  as  accurate  as  the  exact  impressions  made  on 
the  sensitive  plate  by  the  photographic  methods.  But  in  order 
to  secure  the  best  results  with  the  photo-micrographic  apparatus, 
the  sections  must  be  very  thin,  so  that  all  parts  will  be  in  focus 
when  the  picture  is  taken ;  otherwise  an  unpleasant  blur  will 
be  produced.  With  some  kinds  of  objects  it  is  impossible  to 
avoid  the  presence  of  several  planes,  and  the  readjustment  of 
the  microscope  becomes  necessary  in  order  to  bring  out  each 
part  of  the  object  clearly.  Such  specimens  can  only  be  repro- 
duced by  the  camera  lucida.  The  operator  must  be  constantly 
raising  and  lowering  the  objective  while  drawing  the  details  ; 
such  manipulation,  of  course,  would  be  out  of  the  question  while 
using  the  photo-micrographic  camera.  The  student  must,  there- 
fore, learn  to  use  both  the  camera  lucida  and  photo-micrographic 
outfit. 

The  necessary  apparatus  required  for  drawing  the  image  con- 
sists of  a  compound  microscope,  a  camera  lucida,  stage  and 

132 


HOW  TO   MAKE  DRAWINGS  OF  SECTIONS  OF  TISSUE      133 

eyepiece  micrometers,  liquid  India  ink,  sharp-pointed  steel 
pens,  and  good  lead  pencils.  Bernhard's  drawing-desk,  or  Bausch 
&  Lomb's  modification,  may  also  be  added  as  a  convenient  aid, 
though  not  absolutely  necessary. 

The  camera  lucida.  —  This  instrument  is  attached  to  the 
ocular  end  of  the  microscope  and  is  used  for  directing  the  rays, 
which  emanate  from  the  object,  in  such  a  manner  as  to  cause 
the  virtual  image  to  appear  projected  on  the  table  at  the  foot  of 

H 


FIG.  66.  —  Abbe's  Drawing  Camera  Lucida. 

the  microscope,  where  it  may  be  drawn  with  a  pencil.  The  fields 
of  view  on  the  drawing-board,  and  on  the  stage  of  the  micro- 
scope, are  both  combined  in  one  eye,  and  the  image  on  the 
drawing-board  is  made  visible  by  double  reflection.  Two  of  the 
best  camera  lucidas  in  the  market  are  those  made  by  the  Zeiss 
Optical  Company,  of  Germany,  and  Bausch  &  Lomb  Optical 
Company,  of  Rochester,  N.Y.  These  are  shown  in  the  cuts 
(Figs.  66  and  68).  "  The  drawing-surface  is  made  visible  by 
successive  reflections  at  a  large  plane  mirror  and  the  silvered 


134 


BIOLOGICAL  LABORATORY  METHODS 


surface  of  a  small  prism  in  the  eye-point  of  the  eyepiece.  The 
microscopic  image  is  seen  directly  through  an  aperture  in  the 
silvering  of  the  prism,  to  which  is  cemented  another  prism,  both 
prisms  together  forming  a  cube.  Thus  the  pencils  of  light 
reached  the  eye  coincidentally  from  both  the  microscope  and  the 
paper,  and  the  image  and  pencil  are  seen  without  straining  the 
eyes."  1  The  rays  from  the  drawing-surface  first  pass  through 
two  tinted  glasses  of  different  degrees  of  color,  which  serve  to 
equalize  the  illumination  of  the  field  and  paper,  and  they  will  be 
found  necessary  when  high  powers  are  used  for  drawing.  The 


w- 


FlG.  67.  —  Section,  Abbe's  Camera  Lucida. 

arm  upon  which  the  mirror  is  poised  (Fig.  67)  is  10.5  cm.  in 
length,  and  it  is  not  required  to  raise  the  surface  of  the  drawing- 
board  unless  in  the  case  of  very  large  drawings.  This  length- 
ening of  the  mirror  arm  greatly  lessens  the  tendency  to  distortion. 
If  the  drawing  is  large,  it  will  be  necessary  to  raise  or  incline 
the  surface  containing  the  paper.  The  camera  lucida  illustrated 
in  Fig.  66  shows  a  material  improvement  by  Abbe,  in  substi- 
tuting for  the  tinted  glasses  a  movable  cylindrical  cap  jR,  in  the 
wall  of  which  is  placed  a  series  of  tinted  glasses,  and  by  turning 
the  cap  on  its  upper  edge  until  a  small  pin  engages  in  a  corre- 
sponding small  hole  on  the  lower  edge  of  the  cylinder,  each 
smoked  glass  may,  in  turn,  be  brought  in  the  path  of  the  rays 
from  the  drawing-surface.  In  the  cylinder  are  five  tinted  glasses 
of  different  strengths,  while  the  sixth  hole  is  left  empty. 
1  Zeiss*  "  Microscopes  and  Microscopical  Accessories,"  p.  82. 


(  1/NIVERSfTY 

X  C        °F  S 

HOW  TO   MAKE  DRAWINGS  OF  SECTIONS  OF  TISSUE      135 


The  Bausch  Lomb  Optical  Company's  camera  lucida  (Fig.  68) 
is  made  after  Abbe's  pattern,  but  differing  from  the  German 
instrument  in  the  following  particulars  :  — 

1.  The  mirror  arm  is  adjustable  so  that  the  distance  between 
the  mirror  and  the  prism  may  be  increased  or  diminished  at 
pleasure. 

2.  The  mirror  is  extra  large,  thus  increasing  the  surface  of 
reflection  and  correspondingly  giving  a  wider  range  on  the  draw- 
ing-board for  drawing  the  image. 

3.  A  second  series  of  tinted  glasses  are  arranged  to  modify 
the  light  coming  from  the  mirror.     With  the  two  series  of  colored 


FIG.  68.  —  Camera  Lucida  —  Bausch  &  Lomb. 


glasses  it  is  possible  to  secure  a  clear  view  of  the  object  with  the 
combination  of  any  objective  and  ocular. 

For  diminishing  the  brightness  of  the  image  a  disk  B,  as  in 
Bernhard's  apparatus  (Fig.  69),  with  four  smoked  glasses  and  a 
vacant  space,  is  interposed  between  the  prism  and  the  eyepiece. 
In  order  to  pass  conveniently  from  observing  the  image  pro- 
jected on  the  drawing-board  to  observation  directly  through 
the  ocular,  the  instrument  is  so  constructed  that  the  prism  and 
the  diaphragm  may  be  turned  on  the  pivot  Z  to  one  side  and  the 
microscope  can  be  used  as  though  the  camera  is  not  clamped  to 


136 


BIOLOGICAL  LABORATORY  METHODS 


the  tube.  The  eye-circle  is  accurately  centred  with  the  aperture 
in  the  prism  by  means  of  the  two  screws  H  and  L.  When  this 
is  completed,  there  should  be  no  obstruction  to  the  vision  through 
the  ocular  field.  Provision  is  also  made  in  the  instrument  for  a 
spectacle-glass,  in  order  to  render  the  drawing-surface  more  dis- 
tinctly visible.  This  fits  in  the  recess  in  the  top  of  the  cap  R. 
It  is  recommended  by  the  manufacturers  that  in  order  "  to  obtain 
a  fairly  large  field,  drawings  free  from  distortions,  a  slanting  or 


FIG.  69.  —  Bernhard's  Drawing-table. 


raised  drawing-surface  should  be  used."  The  drawing-desk 
devised  by  Dr.  W.  Bernhard  is  most  admirable  for  this  purpose. 
The  following  points  in  its  construction  make  it  particularly  well 
adapted  for  its  purpose  :  The  drawing-board  has  an  exactly 
worked  brass  groove  on  the  upper  plate,  so  that  any  shifting  of 
the  parts  is  prevented.  The  height  and  inclination  of  the  board 
are  controlled  by  means  of  a  graduated  arc.  There  is  an  ad- 
justable hand-rest  provided,  and  it  is  hinged  to  the  drawing- 
plate.  A  supporting  piece  is  hinged  beneath  this  hand-rest,  which 


HOW  TO   MAKE  DRAWINGS  OF   SECTIONS   OF  TISSUE       137 

is  capable  of  extension.  The  entire  apparatus  can  be  inclined 
from  the  plane  of  the  table  upon  which  it  is  resting,  by  means  of 
hinge  connections  with  a  solid  base-plate.  This  is  shown  in  the 
cut.  "  The  plane  of  the  drawing  must  be  at  the  normal  distance 
of  distinct  vision,  i.e.  250  mm.  from  the  eye  of  the  observer, 
since  in  general  the  drawing  should  correspond  in  its  dimensions 


FIG.  70.  — B.  &  L.  Drawing-table— Bernhard's  Modification. 

with  the  microscopic  magnification',  and  therefore  also  the  draw- 
ing-desk must  be  adjustable  in  height  and  inclination  to  the 
microscope."  * 

In  the  drawing  it  is  generally  customary  to  mark  off  only  the 
outlines  of  the  image  on  the  paper,  and  the  details  are  afterward 
put  in  without  the  aid  of  the  camera  lucida.  The  size  of  the 

1  Zeitschr.f.  wiss.  Mikr.,  Vol.  XI,  p.  439,  1892,  and  Vol.  XI,  p.  298,  1894.  Also 
Jour.  Roy.  Mic.  Soc.,  p.  782,  1893. 


138  BIOLOGICAL  LABORATORY   METHODS 

drawing  is  determined  by  the  aid  of  a  micrometer  located  in  the 
ocular.  Before  beginning  the  drawings  the  ocular  micrometer 
must  be  carefully  compared  with  a  stage  micrometer,  in  order 
to  determine  its  exact  value,  and  it  will  be  advisable  to  construct 
a  table  of  known  standard  measures  for  each  of  the  objectives, 
and  then  the  stage  micrometer  may  be  placed  aside  and  the 
ocular  micrometer  used  instead.  When  the  paper  is  put  in 
position  on  the  drawing-board  for  reproducing  the  image,  and 
the  camera  lucida  has  been  adjusted  in  its  place,  the  measure- 
ments should  be  marked  on  the  margin  of  the  drawing  to  show 
the  degree  of  magnification  of  the  image.  When  the  work  is 
finished  in  ink,  this  measurement  should  also  be  permanently 
marked  on  the  paper.  In  work  of  a  high  grade,  this  indication 
of  the  magnification  is  absolutely  necessary,  and  should  always 
be  shown  on  the  drawings  to  make  them  of  scientific  value.  The 
simplest  way  of  securing  this  scale  is  to  place  the  stage  microm- 
eter on  the  microscope  and  draw  its  lines  through  the  camera 
lucida  on  the  margin  of  the  drawing.  Put  the  object  in  the  place 
of  the  stage  micrometer  and  proceed  to  draw  it  in  its  proper  place 
on  the  paper.  Now  in  order  to  determine  how  great  the  enlarge- 
ment is,  apply  to  the  drawing  of  the  image  of  the  object  a  milli- 
meter rule  and  note  how  many  of  the  spaces  on  the  scale  drawn 
on  the  paper  are  covered  by  this  measure  of  the  image,  and  the 
magnification  will  be  obtained  by  dividing  this  number  on  the 
millimeter  rule  by  the  known  value  indicated  on  the  stage  microm- 
eter. Thus  suppose  the  drawing  of  the  image  covers  3  spaces  on 
the  scale  made  on  the  paper,  and  these  3  spaces  measure  45  milli- 
meters on  our  rule,  then  we  wjll  have  45  -r-  .03  =  1500  diameters. 
The  stage  micrometer  is  divided  into  hundredths  of  millime- 
ters, and,  therefore,  the  3  spaces  on  our  drawing  would  represent 
.03  mm.  on  the  micrometer.  If  we  are  using  the  micrometer 
ocular  in  the  work  before  us,  we  must  proceed  as  follows  :  First 
secure  the  ratio  existing  between  the  stage  and  the  ocular  mi- 
crometers, and  then  continue  the  calculations  given  above.  To 
obtain  this  ratio,  place  the  two  micrometers  in  their  proper  posi- 
tions on  the  microscope  and  note  how  many  divisions  on  the 


HOW  TO   MAKE  DRAWINGS  OF  SECTIONS  OF  TISSUE      139 

stage  micrometer  are  covered  by  those  on  the  ocular  micrometer, 
and  then  our  rule  will  be  :  Divide  the  number  given  by  the  stage 
micrometer  by  the  number  given  by  the  ocular  micrometer.  To 
illustrate,  suppose  5  divisions  of  the  stage  scale  correspond  to 
15  divisions  on  the  ocular  scale,  then  the  ratio  will  be :  — 

-r|=-  °°33  +  mm. 

Now  having  obtained  this  ratio  for  the  objective  and  ocular 
in  use  (and  this  must  be  done  for  each  combination  of  the  objec- 
tive and  ocular),  the  size  of  the  object  can  be  found  by  dividing 
the  number  of  divisions  on  the  ocular  micrometer,  covered  by 
the  image  of  the  object,  by  the  ratio  given  for  the  two  microm- 
eters. Thus,  if  the  image  covers  8  divisions  on  the  ocular 
scale,  the  magnification  will  be  :  — 

.08 

=  .024  +  mm.,  or  24  u. 

.0033 

In  drawing  the  object  thus  indicated,  great  advantage  accrues 
to  the  student  in  making  him  careful  about  details  and  accurate 
in  regard  to  results.  He  will  find  that  a  poor  mount  cannot  be 
well  drawn,  and  he  will,  therefore,  be  careful  from  the  start  to 
make  all  his  work  the  very  best  possible.  In  doing  this  drawing, 
the  following  items  may  be  kept  well  in  mind  :  — 

1.  Use  hard  pencils,  with  fine,  sharp  points. 

2.  The  drawing  can  be  best  made  on  Bristol  board,  or  similar 
smooth-surfaced  paper. 

3.  The  light  which  comes  through  the  tube  of  the  microscope 
from  the  object,  and  also  those  rays  which  come  from  the  image 
and  are  reflected  by  the  mirror  of  the  camera  lucida  to  the  eye 
of  the  observer,  should  be  of  nearly  the  same  intensity,  in  order 
to  bring  out  clearly  both  the  point  of  the  pencil  and  the  image. 
There  are  several  ways  of  regulating  the  intensity  of  the  light  at 
each  point.     If  the  stage  is  too  bright,  the  Abbe  sub-stage  illu- 
minator will  readily  correct  the  trouble.     If  the  microscope  is 
not  provided  with   the  Abbe  illuminator,  then  the  diaphragm 
must  be  used  with  a  smaller  opening,  or  a  screen  must  be  placed 
between  the  stage  and   the  source  of   light.     To   know  which 


140  BIOLOGICAL   LABORATORY   METHODS 

field  is  the  brightest,  the  student  must  watch  the  image  of  the 
pencil  and  of  the  object,  and  if  the  latter  is  the  most  distinct, 
then  he  will  at  once  know  that  the  stage  of  the  microscope  is  too 
bright ;  if  the  point  of  the  pencil  is  the  most  distinct,  then  the 
surface  of  the  paper  has  too  much  light  on  it.  The  character  of 
light  coming  from  the  surface  of  the  paper  can  be  controlled  by 
the  camera  lucida,  if  Zeiss's  pattern  is  used,  as  has  already  been 
indicated.  This,  however,  may  be  accomplished  also  by  placing 
a  screen  of  ground  glass  between  the  source  of  light  and  the 
drawing-board.  These  mechanical  devices,  however,  for  control- 
ling the  quantity  of  light  can  be  multiplied  by  the  ingenuity  of 
the  student. 

4.  Fasten  the  paper  securely  to  the  drawing-board,  so  that  it 
will  not  be  displaced  during  the  operations. 

5.  Follow  faithfully  the  lines  of  the  image,  taking  care  not  to 
make  heavy  markings  with  the  pencil.     Make  all  cell  walls,  of 
an  appreciable  thickness,  with  double  lines,  and  the  solid  por- 
tions of  the  sections  should  be  well  shaded. 

6.  Note  on  all  drawings  not  only  the  magnification,  but  also 
as  well  the  number  or  letter  of  the  objective  and  ocular.     This 
is  not  often  done  on  drawings,  but  it  is  very  desirable  when 
work  of  a  high  grade  is  performed,  in  order  that  satisfactory 
comparisons  may  be  made  at  any  time. 

7.  If  the  outlines  of  the  image  are  made  with  a  low  power, 
and  the  details  are  put  in  with  a  higher  power,  this  fact  must  be 
clearly  indicated  on  the  drawing. 

8.  The  eye  must  be  held  close  to  the  microscope,  and  look 
perpendicularly  down,  so  as  not  to  distort  the  image  and  produce 
an  incorrect  drawing. 

9.  The  height  of  the  drawing-desk  must   be  lowered  or  ele- 
vated until  its  surface  is  brought  into  the  distinct  visual  distance 
of  the  observer.     This  height  can  be  determined  by  the  distinct- 
ness of  the  inclined  surface  of  the  desk.     Draw  the  measure- 
ments of  a  stage  micrometer  on  the  paper,  and  if  the  lines  of  the 
divisions  stand  at  unequal  distances,  separating  more  and  more, 
as  you  approach  the  top  of  the  desk,  the  surface  needs  inclining. 


CHAPTER   IX 

PHOTO-MICROGRAPHS.       THE   APPARATUS 

THE  remarkable  strides  made  in  the  science  of  photography 
within  the  past  few  years  have  rendered  it  possible  to  reproduce 
with  great  exactness  the  image  of  the  object,  and  with  such  a 
degree  of  facility  as  to  place  the  results  within  the  reach  of  any 
microscopist  who  possesses  a  general  knowledge  of  chemical  laws, 
and  who  will  exercise  the  ordinary  degree  of  care  in  the  use  of 
delicate  apparatus  and  chemicals. 

Before  entering  upon  a  discussion  of  the  methods  used  for 
making  the  negatives  and  positives,  it  may  be  best  to  give  a 
detailed  account  of  the  apparatus  and  chemicals  required  for  a 
satisfactory  prosecution  of  the  work.  In  the  descriptions  which 
follow,  it  is  assumed  that  the  student  is  a  novice  in  the  science 
of  photography,  and  therefore  the  subject  is  treated  fully  in 
order  to  make  the  methods  clearer  to  the  minds  of  the  beginners. 

As  in  the  case  of  the  microscope  and  its  accessories,  so  is  it 
true  in  reference  to  the  photographic  outfit :  the  best  apparatus 
made  by  the  most  skilful  wrorkmen  will  be  the  cheapest  in  the 
end.  A  lens  made  to  sell  and  offered  below  what  is  a  reason- 
able price  for  a  first-rate  glass  will  soon  be  found  inadequate  to 
meet  the  demands,  and  only  inferior  pictures  will  be  the  results. 

The  camera.  —  The  following  sizes  of  view  cameras  will  be 
found  very  convenient  and  useful  in  the  laboratory :  4  x  5  and 
6J  x  8^.  The  different  sizes  of  kits  should  be  purchased  with 
the  cameras,  so  that  the  several  sizes  of  negatives  may  be  made 
without  requiring  the  purchase  of  other  cameras.  It  will  be 
possible  to  do  all  needed  work  with  the  cameras  above-men- 
tioned, but  in  a  well-equipped  laboratory  it  will  be  found  desir- 
able for  convenience  to  add  also  a  photo-micrographic  camera, 

141 


142  BIOLOGICAL  LABORATORY   METHODS 

either  Bausch  &  Lomb  Optical  Company's  or  Zeiss's  pattern. 
These  last  are  very  expensive,  but  the  laboratory  that  contains 
one  of  these  outfits  for  photo-micrographic  work  is  prepared  to 
accomplish  high-grade  results  with  the  least  degree  of  incon- 
venience. 

As  previously  stated,  two  sizes  of  cameras  will  be  sufficient 
for  most  of  the  photographing  required  in  the  laboratory.  The 
4X5  camera  may  be  of  the  Kodak  style  or  the  Premo  pattern ; 
but  this  apparatus  should  have  an  extra  length  of  bellows,  so 


FIG.  71.  —  Folding  Kodak  Camera. 

that  the  instrument  can  be  used  for  many  purposes  requiring  a 
long  bellows.  There  are  several  good  hand-cameras  on  the 
market,  like  the  Kodak  and  Premo,  any  one  of  which  may  be 
used  instead  of  the  two  mentioned,  provided  the  essentials  found 
in  these  two  excellent  instruments  are  secured. 

In  the  following  description  of  the  hand-camera  the  cartridge 
kodak  is  taken  as  the  pattern  of  the  type  of  these  convenient 
instruments,  not  that  it  is  considered  by  the  author  to  be  the 
best,  but  because  it  has  some  additions  which  are  not  found  in 
others. 


PHOTO-MICROGRAPHS.      THE  APPARATUS 


143 


The  kodak  may  be  used  as  a  hand-camera  (Fig.  7 1),  or  it  may 
be  mounted  on  a  tripod  when  exact  focussing  is  desired.     The 

meaning  of  the  term  "  hand-camera  " 
is  in  reference  to  those  instruments 
which  are  so  constructed  that  they 
can  be  held  in  the  hands  when  used 
in  taking  "  snap-shots,"  or  pictures  of 
objects  which  are  moving,  or  of  bright 
objects  when  the  exposures  are  made 
as  short  as  -^  of  a  second,  or  shorter. 
It  is  wise  to  purchase  with  the  camera 
a  tripod,  in  order  that  delicate  focus- 
sing may  be  done  at  any  time  desired. 
This  is  a  support  for  the  camera,  so 
constructed  that  it  may  be  folded  up 


FIG.  72. —  Camera  Tripod. 


FIG.  73.  —  Carrying-case  for  Tripod. 


when  on  a  trip,  and  stored  away  in  the  carrying-case  (Fig.  73). 
It  will  always  pay  to  place  the  camera  on  the  tripod  and  care- 
fully focus  the  picture  on  the  ground  glass,  if  the  object  to  be 
photographed  will  permit  of  such  treatment,  because  it  is  almost 
impossible  to  secure  sharp  photographs  by  holding  the  camera 
in  the  hands  and  snapping  at  objects,  the  distance  being  only 
guessed  at.  In  some  instances,  however,  the  tripod  cannot  be 
used,  and  then  the  value  of  the  instrument  as  a  hand-camera 
becomes  quite  evident. 

There  is  always  attached  to  these  cameras  a  "  view  finder," 
which  is  a  small  lens  reproducing  in  miniature,  under  the  eye  of 
the  photographer,  a  picture  of  the  object  desired  on  the  sensi- 
tive plate.  This  view  finder  enables  the  photographer  to  watch 
the  moving  object,  and  just  as  it  moves  across  the  field  of  the 
instrument  he  can  snap  the  shutter  in  front  of  the  lens  and  take 
the  picture. 


144  BIOLOGICAL  LABORATORY   METHODS 

At  the  rear  of  the  camera  is  located  the  ground-glass  plate,  on 
which  to  focus  the  picture  of  the  object  when  the  tripod  is  used 
and  time  exposures  are  made.  The  bellows  allows  the  front  and 
rear  of  the  instrument  to  be  adjusted  so  that  focussing  may 
be  accomplished,  and  the  camera  may  be  folded  up  and  placed 
in  the  carrying-case  during  transportation  and  to  exclude  dust 
when  the  instrument  is  not  in  use.  The  hand-cameras  now 
made  are  constructed  to  fold  within  themselves  into  a  compact 
form,  and,  being  covered  with  neat  leather,  they  do  not  require 
a  case. 


FIG.  74.  —  Roll-holders. 

In  connection  with  these  hand-cameras  roll-holders  are  used 
(Fig.  74),  in  which  a  roll  of  thin  celluloid  highly  sensitized  is 
placed,  and  so  mounted  in  the  holder  as  to  permit  of  being 
brought  by  the  mechanism,  a  portion  at  a  time,  before  the  lens 
to  receive  the  picture.  Each  roll  of  film  contains  six  or  twelve 
4x5  inch  surfaces,  so  that  as  many  as  six  or  twelve  pictures 
may  be  impressed  on  the  sensitive  surface  of  each  roll  before 
reloading  the  holder  becomes  necessary.  These  cartridges,  or 
rolls,  are  so  constructed  that  the  loading  of  the  holder  may  be 
done  anywhere  and  in  the  brightest  light.  The  film  is  put  up  in 
light-tight  rolls,  as  shown  in  the  illustration  (Fig.  75).  Extending 


PHOTO-MICROGRAPHS.      THE  APPARATUS  145 

the  full  length  of  the  spool  and  several  inches  beyond  each 
,end  of  the  sensitive  film  are  strips  of  black  paper.  This  paper 
is  also  continuous  back  of  the  entire  length  of  the  film.  When 
the  roll  of  film  is  inserted  in  the  holder,  the  end  of  the  black 
paper  is  attached  to  the  extra  roll  and  the  holder  is  closed,  so 
that  all  white  light  is  excluded.  The  key  is  now  turned  until 
the  sensitive  film  covers  the  space  behind  the  lens,  when  the 
exposure  is  made  for  the  first  picture.  At  .«<?  s^-^v 

proper  intervals  on  the  back  of  the  black 
paper,  and  behind  the  film,  are  marked  in 
white  the  figures  i,  2,  3,  4,  etc.,  represent-  j 
ing  the  number  of  exposures  possible  on 
the  sensitive  surface  of  the  film.  These 
numbers  are  visible  through  a  small  open- 
ing in  the  back  of  the  holder,  which  is 
covered  with  red  celluloid.  When  the  last 
exposure  is  made,  a  few  more  turns  are  '; 
given  to  the  key,  so  that  the  extra  length 
of  black  paper  may  be  also  wrapped  on 

r  FIG.  75.  —  Roll  of  Film. 

the  roller  and  cover  the  sensitive  surface 

of  the  celluloid,  and  then  the  roll  is  taken  out  and  a  fresh  roll  is 
inserted  in  its  place  in  the  holder.  All  of  this  may  be  done  in 
white  light,  if  care  is  exercised  to  manipulate  the  black  paper 
properly  at  the  end  of  the  sensitive  film.  The  best  hand- 
cameras  in  the  market  are  constructed  for  use  with  the  roll- 
holder,  and  they  also'  have  attached  facilities  for  the  use  of 
sensitive  glass  plates  and  cut  films. 

The  larger  camera,  of  6£  x  8|  inches,  may  be  any  make, 
provided  it  is  manufactured  out  of  the  best  materials.  There 
are  a  number  of  good  instruments  on  the  market  which  will 
subserve  all  the  purposes  demanded  in  the  laboratory,  and  they 
must  possess  the  following  important  properties  :  — 

1.  It  must  have  a  double  rising  front  for  lowering  or  elevating 
the  lens  without  disturbing  the  position  of  the  tripod.      This 
gives  wide  range  of  adjustment. 

2.  It  must  have  a  double  swing  back,  which  will  insure  a 

L 


146       BIOLOGICAL  LABORATORY  METHODS 

clear,  sharp  focus  on  the  object ;  it  makes  no  difference  how  the 
object  may  be  placed  in  front  of  the  camera. 

3.  There  must  be  a  rack  and  pinion  adjustment,  so  that  the 
ground  glass  will  remain  stationary  while  the  focussing  is  made 
on  the  glass. 

4.  The  ground-glass  frame  should  be  hinged  to  the  camera, 
so  that  it  will  not  be  broken  during  the  time  of  exposure  by 
some  one  stepping  on  it  if  laid  on  the  floor  or  ground. 

The  Lens. — Two  sizes  of  the  photographic  lens  will  be  found 
sufficient  for  the  usual  work  in  the  laboratory,  —  4  x  5  and 
6|-  x  8^-.  A  wide-angle  lens  will  also  be  found  very  useful  in 
certain  kinds  of  work,  and  it  will  be  well  also  to  purchase  one 
of  these  for  the  laboratory.  The  following  lenses  are  considered 
to  be  among  the  best  now  offered  in  the  market :  — 

Alvan  G.  Clark's  Lens.  Morrison's  Lens. 

Beck's  Autpgraph.  Queen's  Pantagraph. 

Dallmeyer's  Lens.  Ross's  Lens. 

Darlot's  Lens.  Steinheil's  Aplanatic  Lens. 
Francais's  Extra  Rapid  Rectilinear.       Suter's  Aplanatic. 

Goerz's  Double  Anastigmat.  Voiglander's  Lens. 

Gundlach's  Lens.  Zeiss's  Anastigmat. 

The  above  list  may  be  classified  as  follows,  according  to 
merit :  — 

1.  Zeiss's  Anastigmats.  C  Alvan  G.  Clark's  Lens. 

2.  Dallmeyer's  Lens.  J   Gundlach's  Lens. 

{Beck's  Autograph.  |   Voiglander's  Lens. 

Goerz's  Double  Anastigmat.  L  Steinheil's  Aplanatic. 

Suter's  Aplanatic. 

5.    The  others  mentioned  in  the  list  may  be  placed  under  this  head. 

In  the  selection  of  a  photographic  lens,  the  following  requisites 
must  be  carefully  noted :  — 

1.  Achromatism,  of  course,  is  of  first  consideration,  and  the 
lens  must  stand  the  test  well,  because  a  slight  deviation  from 
achromatism  will  produce  an  indistinct  image. 

2.  Distortion  of  the  image  on  the  outer  limits  must  be  re- 
duced to  the  least  degree  possible. 


PHOTO-MICROGRAPHS.      THE  APPARATUS  147 

3.  Rapidity  in  action  and  rectilinearity  in  results  are  absolutely 
required  in  all  first-class  lenses.     The  rapidity  of  a  lens  depends 
upon  the  size  of  the  stops  in  the  diaphragm.     The  larger  the 
opening,  more  light  can  enter  and  quicker  will  be  the  action  on 
the  sensitive  plate.     The  size  of  the  diaphragm  is  controlled  by 
the  focal  length  of  the  lens  ;  that   is  to  say,  if  aperture  f  16 
be  used,  it  signifies  that  the  size  of  the  beam  of  light  which 
enters  the  lens  is  one-sixteenth  of  the  focal  length.     This  prop- 
erty of   the   objective  has  been  carefully  worked   out   by  the 
Photographic  Society  of  Great  Britain,  as  follows  :  — 

Aperture :    f  4-f  5.6-f  8-f  I  i-3-f  i6-f  22.6-f  32-f  45-3-f  64. 
Relative  exposure  ratio  :    1-2-4-8-16-32-64-128-256. 

By  the  adoption  of  this  series  of  apertures  one  lens  can  be 
compared  with  another.  In  the  use  of  the  stops  it  must  be 
remembered  that  each  requires  twice  the  exposure  of  the  one 
preceding  to  produce  the  same  results  on  the  sensitive  plate. 
The  special  advantages  in  their  use,  however,  consist  in  reduc- 
ing to  a  minimum  the  blurring  of  the  image  on  the.  outer  mar- 
gins, and  in  producing  sharpness  in  detail  which  is  especially 
pleasing  in  the  picture.  The  small  stops  also  increase  the 
depths  of  definition. 

4.  The  angle  of  view  should  not  be  less  than  50°  for  the  rec- 
tilinear type,  while  the  wide-angle  series  should  cover  an  angle 
of  not  less  than  85°. 

5.  Depth  of  focus   depends  on  the  angle   of  view,  working 
aperture,  and  focal  length.     As  these  increase,  the  depth  of  the 
focus  decreases,  and  vice  versa.     Depth  of  focus  is  due  to  the 
converging  rays  striking  the  plate  at  a  more  acute  angle. 

6.  Flatness  of  image  over  the  entire  surface  of  the  ground- 
glass  plate  by  extending  the  oblique  pencils  of  light. 

7.  Depth  of  definition  to  give  a  sharp,  clearly  defined  image 
on  any  portion  of  the  plate,  by  reducing  spherical  aberration. 
When  the  focus  of  the  lens  is  shortened,  the  greater  will  be 
the  depth  of  definition.     Thus  it  is  that  in  the  case  of  a  short 
focus  lens  there  will  be  projected  on  the  sensitive  plate  a  greater 


148 


BIOLOGICAL   LABORATORY   METHODS 


number  of  well-defined  objects  in  the  foreground  than  will  be 
secured  in  the  use  of  a  lens  of  longer  focus.  This  is  a  valuable 
property  in  landscape  work,  where  distance  comes  in  as  an  im- 
portant factor. 

The  following  addi- 
tional apparatus  will  be 
required  for  making  the 
negatives  and  positives : 
Focussing  cloth  and 
focussing  glass. 

Double  plate-holders. 
Diaphragm  shutter.  — 
One  of  the  best  of  these 
shutters  is  that  made  by 
the  Bausch  &  Lomb  Opti- 
cal Company.  The  illus- 
tration of  this  instrument 
is  given  in  Fig.  76.  In 
speaking  of  this  shutter 
the  manufacturers  have 
the  following  to  say  in 
regard  to  its  merits, 
which  the  author  after 
several  years'  use  of  the 
instrument  can  indorse  : 

"  The  construction   of 
FIG.  76.  — Diaphragm  Shutter.  ......  . 

the  ins  diaphragm  shut- 
ter is  based  on  a  careful  consideration  of  the  optical  principles 
involved,  and  it  is  without  question  the  most  scientifically  cor- 
rect shutter  yet  produced.  It  can  be  easily  proven,  both  theo- 
retically and  by  actual  experience,  that  as  the  size  of  the  stop  is 
decreased,  the  lens  has  not  only  greater  covering  power,  but  the 
illumination  (exposure)  becomes  more  uniform.  Furthermore,  it 
is  well  known  that  exposures  with  large  openings  give  greater  con- 
trast between  the  high  lights  and  shadows,  while  with  small  stops 
and  long  exposures  greater  detail  is  obtained  in  the  shadows. 


PHOTO-MICROGRAPHS.      THE  APPARATUS 


149 


It  has  setting  device  to  give 


For  this  reason  a  shutter  constructed  on  the  iris  diaphragm 
principle,  opening  in  the  centre  with  a  minute  aperture,  and 
gradually  enlarging  to  the  full  opening  desired,  then  gradually 
reducing  its  opening  to  the  closing  point,  gives  the  high  lights 
full  value  and  sharpness,  depth  of  focus  and  detail  in  the  shad- 
ows approaching  the  results  obtained  with  small  stops,  so  that 
in  actual  use  the  diaphragm  shutter  gives  great  covering  power, 
more  equal  illumination,  and  greater  depth  of  focus  than  is  pos- 
sible with  any  other  shutter.  The  following  are  some  of  its 
advantages :  — 

"It  is  placed  between  the  systems  of  the  lens,  at  the  dia- 
phragm point,  thus  acting  as  stop  and  shutter  at  once.  No  extra 
stops  are  needed.  It  gives  absolute  uniform  illumination  over 
the  entire  plate.  It  gives  brilliant  high  lights,  and  at  the  same 
time  definitions  in  the  shadows.  It  gives  automatically  any  ex- 
posure from  one-hundredth  of  a  second  to  three  seconds,  and 
time  exposures  for  any  duration, 
any  size  opening  from  pin- 
hole  to  the  largest  stop. 
It  is  operated  either  with 
pneumatic  bulb  or  finger 
release.  It  cannot  open, 
or  expose  the  plate,  while 
being  set.  It  does  not  jar 
the  camera,  even  when 
working  at  the  highest 
speed.  It  is  especially 
adapted  for  hand-camera 
lenses." 

Ruby  lamp  for  the  dark 
room;  —  This  lamp  may  be 
either  in  the  'form  of  oil 
(Fig.  77),  gas,  or  electric  lamp.  White  light  must  not  be  per- 
mitted to  escape  from  the  lamp  during  the  work  with  the  sensi- 
tive plate  in  the  dark  room  in  developing  the  picture  and  fixing 
it  on  the  surface  of  .the  glass,  or  film,  so  that  exposure  to  white 


FlG.  77.  —  Carbutt's  Multum  in  Parvo 
Ruby  Lamp. 


ISO 


BIOLOGICAL   LABORATORY   METHODS 


light  will  not  destroy  it.  The  lamp  must  be  made  with  a  com- 
bination of  ruby  and  orange  glass,  or  ruby  glass  covered  with 
orange  tissue-paper.  The  light  sifting  through  such  a  protec- 
tion will  "not  affect  ordinary  sixteen-sensitometer  plates,  and  it  is 
safe  for  orthochromatic  plates  and  films,  provided  they  are  not 
exposed  to  it  longer  than  a  few  moments  at  a  time. 

Ray  filter  or  color  screen.  —  In  photographing  microscopical 
objects  which  have  been  colored  with  anilin  or  other  dyes,  it 
becomes  necessary  to  interpose  between  the  sensitive  plate  and 

the  object  a  color 
screen,  which  will 
serve  to  increase 
or  decrease  the 
intensity  of  cer- 
tain colors  of  the 
spectrum.  The 
ray  filter  (Fig.  78) 
is  a  metal  ring 
or  cylinder,  with 
each  end  closed 
by  a  glass  disk. 
In  this  cell  thus 
formed  by  the 
disks  and  cylin- 
der is  placed  the 
colored  solution  for  sifting  the  light  before  it  reaches  the  sensi- 
tive plate.  This  ray  filter  is  made  to  place  on  the  hood  of  the 
photographic  lens  like  a  cap.  The  solution  is  bichromate  of  pot- 
ash. The  effect  of  this  sifting  of  the  colors  of  light  before  they 
reach  the  sensitive  plate  is  to  give  a  picture  which  produces 
true  color  values,  and  renders  correct  gradation  of  shading  and 
sharpness  in  details  throughout  the  entire  photograph.  The 
ray  filter  is  particularly  valuable  for  making  photo-micrographs. 
Developing  trays  of  various  sizes.  —  A  number  of  these  indis- 
pensable vessels  should  be  in  the  dark  room  for  the  various 
purposes  for  which  they  are  made.  They  are  manufactured  out 


FIG.  78.  —  Ray  Filter. 


PHOTO-MICROGRAPHS.      THE  APPARATUS 


of  glass,  rubber,  and  other  material  not 
affected  by  the  chemicals  used  in  the  de- 
veloping of  photographic  plates. 


FIG.  80.  —  Graduate. 


FIG.  79.  —  Developing  Tray. 

Graduates  for  measuring  the  chemicals. 
—  The  following  sizes  will  be  found  to  be 
useful  in  the  laboratory  and  in  the  dark 
room  :  4  ounces,  32  ounces.  (Fig.  80.) 

Fixing-baths  made  of  hard  rubber.  —  The  Gennert  pattern  is 
excellent  for  several  reasons.  They  have  grooves  extending 
from  the  top  to  the  bottom  of  the  vessel  (Fig.  81),  in  which  the 
plates  slide  and  stand  in  a  vertical  position,  thus  preventing 
injury  from  the  precipitates  which  form  in  the  solutions.  A  top 
covers  the  vessel,  so  that  both  dust  and  light  are  excluded  dur- 
ing the  fixing  of  the  plates  in  the  hyposulphite  solution.  There 
are  two  sizes  made,  one  for  plates 
3!  x  4j,  4i  X  61,  and  6%  X  8£,  and 
one  for  plates  4  X  5,  5  X  7,  5  X  8, 
and  8  x  10. 

Negative  washing-bath.  —  The  ves- 
sels made  of  glass  are  more  durable 
and  are  easily  kept  clean.  These 
baths  (Fig.  82)  are  used  for  eliminat- 
ing the  hyposulphite  of  soda  from  the 

negatives  after  they  have  been  fixed 

,         i     t  i  •/•    ,-.  FIG.  8 1.  —  Fixing-bath. 

in    the   dark   room,   because    if   the 

hyposulphite  of  soda  is  left  in  the  film  of  the  negative,  the  pic- 
ture will  soon  be  destroyed  by  the  chemical  action  of  the  soda 
on  the  gelatin  film.  The  plates  are  washed  by  running  water 


152 


BIOLOGICAL   LABORATORY   METHODS 


into  the  bottom  of  the  bath,  where  the  plates  are-  resting  on  their 
edges ;  and  as  the  water  runs  out  of  an  orifice  at  the  top  of  the 
vessel,  it  dissolves  out  the  hyposulphite  of  soda  and  leaves  the 
negative  free  from  that  destroying  agent. 


FIG.  82.  —  Negative  Washing-bath. 

Light-tight  boxes  of  various  sizes  for  holding  the  unexposed 
plates  and  keeping  them  from  white  light. 

Printing-frames  (Fig.  83),  to  agree  with  the  size  of  the  plates 
used  in  the  camera  for  photographing  objects  in  the  laboratory 
or  in  the  field.  The  sizes  usually  found  convenient  are  3 \  x  4^, 
4X5>5X7>5XV8>  and  61  x  8£.  These  frames  should  have 
a  clear  glass  plate  fitted  to  each,  so  that  celluloid  films  from  the 
hand-cameras  may  be  also  used  in  them. 


PHOTO-MICROGRAPHS.      THE  APPARATUS 


153 


Negative  dry- 
ing -  racks.  —  The 
racks  (Fig.  84)  are 
for  the  purpose  of 
drying  the  nega- 
tives after  they 
have  been  thor- 
oughly washed 
and  swabbed  with 
absorbent  cotton 
to  clear  off  the 
particles  adhering 
to  the  surface  of 
the  film.  FIG' 83>  ~  Printins-frame- 

Retouching-frame.  —  Even  under  the  most  careful  manipula- 
tion of  the  photographer,  it  sometimes  becomes  necessary  to 
touch  the  negative  here  and  there  with  a  pencil  or  brush  to 
smooth  over  blemishes.  The 
retouching  -  frame  (Fig.  85)  is 
adapted  to  this  work.  The 
negative  is  placed  on  the 
ground-glass  plate  A,  and  the 
slide  E  is  adjusted  so  as  to 


FIG.  84.  —  Negative  Drying-rack. 


FlG.  85.  —  Retouching-frame. 


hold  the  plate  in  position,  and  the  mirror  C  is  turned  so  that 
the  light  will  reflect  up  through  the  negative  in  order  that  the. 


154  BIOLOGICAL   LABORATORY   METHODS 

operator  may  locate  the  spots  to  be  worked  on ;  the  part  B  is  to 
shade  the  operator's  eyes  from  the  light,  and  the  drawer  D  is 
intended  to  hold  the  pencils  and  brushes  when  not  in  use. 

Camel's-hair  brush,  two  inches  wide,  for  dusting  the  plates 
before  they  are  put  in  the  holders  for  the  camera.  The  necessity 
for  this  dusting  of  the  plates  is  to  prevent  the  formation  of  pin- 
holes  in  the  negative,  caused  by  the  dust  particles  covering  those 
portions  of  the  plate  and  thus  preventing  the  full  action  of  the 
light  on  the  sensitive  surface. 

An  enlarging-easel.  —  This  is  an  important  addition  to  the 
laboratory  outfit  if  any  enlarging  is  done,  that  is,  if  the  operator 
desires  to  make  a  picture  larger  than  can  be  secured  with  any 
one  of  the  cameras  belonging  to  the  laboratory.  A  skilful  car- 
penter can  make  this  easel ;  it  is  only  necessary  to  have  a  vertical 
board  on  which  the  sensitive  paper  may  be  stretched,  and  the 
board,  fastened  to  upright  supports  resting  on  rollers,  so  that  the 
easel  may  be  readily  moved  back  and  forth  to  get  the  desired 
enlargement. 

Exposure  meters.  —  It  is  a  difficult  matter,  especially  with  the 
amateurs  in  photography,  to  know  just  how  long  the  sensitive 
plate  should  be  exposed  to  the  action  of  the  light  in  order  to 
secure  the  best  results.  To  obviate  this  trouble  in  great  measure, 
there  are  a  number  of  exposure  meters,  or  scales,  and  other  de- 
vices on  the  market,  some  of  which  are  excellent,  while  others 
are  indifferent.  Two  of  the  best  of  these  meters  are  given  be- 
low. The  author  has  experimented  with  them  and  has  obtained 
satisfactory  results.  . 

Wager* s  exposure  meter.  —  This  is  an  aluminum  plate,  on  which 
slides  two  ivory  verniers.  Printed  on  the  plate  and  the  verniers 
are  values  for  dates,  hours  of  the  day,  character  of  the  sky  and  the 
objects,  diaphragm  stops,  name  and  speed  of  plates  and  films, 
and  time  of  exposure.  The  correct  exposure  of  plate  is  obtained 
by  knowing  the  speed  of  the  plate,  the  hour  in  the  day,  the  sea- 
son of  the  year,  and  the  size  of  the  diaphragm.  By  moving  the 
ivory  slides  to  the  correct  positions  on  the  aluminum  plate,  the 
time  of  exposure  in  seconds  and  in  minutes  is  read  off  at  once. 


PHOTO-MICROGRAPHS.      THE  APPARATUS  155 

Watkins'  exposure  meter.  —  This  instrument  has  also  the 
factors  mentioned  as  belonging  to  the  meter  above,  but  the 
correct  time  of  exposure  is  obtained  by  exposing  to  the  action 
of  the  light  a  narrow  strip  of  sensitive  paper,  while  seconds  are 
counted  by  the  vibration  of  a  pendulum  attached  to  the  instru- 
ment. The  movement  of  the  verniers  on  this  meter  will  give 
the  time  of  exposure  for  the  plate. 

The  necessary  chemicals  demanded  in  the  photographic  ma- 
nipulations will  be  considered  when  the  formulae  are  given  in 
describing  the  methods  for  making  the  negatives  and  positives. 
But  it  must  be  borne  in  mind  that  the  purest  chemicals  should 
be  purchased  and  used  in  all  photographic  operations.  Inferior 
and  unsatisfactory  results  will  meet  the  student  unless  he  ob- 
serves rigidly  this  rule. 


CHAPTER   X 

MAKING   THE    PHOTO-MICROGRAPHS   (CONTINUED) 

Arranging  and  adjusting  the  apparatus.  —  Some  place  in  the 
laboratory  must  be  found  where  there  is  the  least  degree  of 
vibration,  caused  bypassing  to  and  fro  in  the  building  and  from 
outside  influences,  and  there  the  table  containing  the  apparatus 
for  focussing  and  exposing  the  sensitive  plate  must  be  located. 
If  the  building  has  a  well  lighted  and  ventilated  basement,  with 
a  cemented  floor,  no  better  place  can  be  secured  for  performing 
all  the  work  in  photo-micrography.  It  must  be  remembered, 
however,  that  wherever  the  apparatus  is  located,  decided  vibra- 
tions given  to  the  table  will  sadly  spoil  all  results  by  producing 
a  blurred  picture.  All  parts  of  the  apparatus  used  for  holding 
the  slide,  the  camera,  and  focussing  appliances,  and.  the  con- 
densers for  concentrating  and  controlling  the  distribution  of  the 
light,  should  all  be  fastened  to  the  same  rigid  base,  so  that  if 
slight  vibrations  occur  in  the  room,  the  entire  outfit  will  move  as 
a  whole,  and  there  will  be  less  tendency  to  blurring  the  negative. 

Since  the  temperature  of  the  atmosphere  influences,  to  a  very 
large  degree,  the  results  secured  in  photographing,  it  will  be  of 
great  advantage  to  have  the  room  under  perfect  control,  so  that 
the  temperature  may  be  lowered  and  raised  at  pleasure.  It  has 
been  found,  from  experience,  that  the  best  negatives  are  made 
when  the  temperature  of  the  chemicals  range  from  15°  to  21°  C. 
If  these  solutions  used  in  developing  are  not  kept  within  this 
range,  the  negatives  will  be  unsatisfactory.  Cold  retards  the 
results,  while  heat  accelerates  and  causes  flatness.  In  summer, 
therefore,  ice  should  be  kept  around  the  vessels  containing  the 
developing  solutions  until  the  temperature  is  lowered  to  the  re- 
quired degree,  and  the  dark  room  must  also  be  regulated,  as  far 
as  possible,  to  reach  these  same  results. 

156 


MAKING  THE   PHOTO-MICROGRAPHS  157 

Although  there  are  several  excellent  brands  of  dry  plates  on 
the  market,  all  giving  good  results  in  the  hands  of  the  careful 
manipulator,  still,  it  will  be  wise  to  study  the  qualities  of  the 
plates  made  by  one  manufacturer  and  use  them  in  most,  if  not 
all,  experiments  conducted  in  the  laboratory.  In  determining 
this  question,  careful  notes  should  be  taken  of  each  exposure, 
and  also  the  action  of  the  developing  fluids  on  the  plates.  By 
this  method  all  of  the  capabilities  of  the  plates  will  be  fully 
understood.  The  experience  of  the  author  has  convinced  him 
that,  for  general  scientific  work,  Carbutt's  plates  are  superior  to 
most  of  the  brands  sold  in  America.  This  conclusion,  however, 
may  be  due  to  the  fact  that  he  is  more  familiar  with  these  plates 
than  with  the  others  ;  but,  as  has  been  already  stated,  the  same 
amount  of  experience  with  the  others  may  yield  as  good  results. 
Carbutt's  plates  give  brilliant  negatives,  and  they  allow  con- 
siderable latitude  in  exposure  and  development.  The  following 
represent  the  brands  he  manufactures  which  are  suitable  for  the 
experiments  in  the  laboratory  :  — 

"  Eclipse,  sensitometer  23  and  27.  —  Films  and  plates  are  ex- 
tremely sensitive,  and  specially  intended  for  quick  studio  expos- 
ures, concealed  and  detective  cameras,  instantaneous  views, 
and  magnesium  flash-light  photography. 

"  B  plates,  sensitometer  16  to  20.  —  For  landscape  views  and 
general  photography. 

"  Orthochromatic  plates,  sensitometer  23  to  27.  —  Give  cor- 
rect color  values  when  exposed  through  ray  filters  or  color 
screens.  The  best  plates  for  landscapes,  interiors,  and  photo- 
micrography. 

"  Polychromatic  plates.  —  Color  sensitive  to  entire  spectrum. 

"  Non-halation  plates  with  orthochromatic  quality.  —  These 
plates  are  suitable  for  subjects  that  transmit  much  light,  like 
windows  and  bright  polished  metal  surfaces. 

"  Cut  films  are  also  manufactured  by  Carbutt,  containing  the 
properties  described  in  the  above  glass  plates. 

"  Process  plates  make  good  photo-micrographs,  producing 
clear,  sharp  detail  and  abundant  contrast. 


158  BIOLOGICAL  LABORATORY  METHODS 

"  Lantern  plates.  —  Giving  results  of  great  brilliancy  and  fine 
color.  For  lantern  slides  and  window  transparencies.  The 
sensitive  film  is  mounted  either  on  glass  plates  or  celluloid. 

"  The  sensitometer  number  on  each  box  of  plates  indicates 
their  sensitiveness ;  the  higher  the  number,  the  greater  the 
rapidity  of  the  plate.  For  portraiture  on  the  special  and  eclipse 
plates  under  the  skylight,  if  an  eclipse  sensitometer  27  required 
2  seconds,  a  special  sensitometer  27  would  require  3^-  seconds, 
and  a  special  sensitometer  24,  4§  seconds."  (Carbutt.) 

The  following  firms  also  make  standard  plates,  which  are 
highly  commended  by  both  professional  and  amateur  workers, 
and,  as  was  stated  on  a  previous  page  of  this  book,  the  student 
should  find  the  brand  of  plate  which  suits  his  experiment,  be- 
come familiar  with  the  qualities  of  that  brand,  and  do  most  if 
not  all  of  his  work  with  it.  Trying  first  one  plate  after  another 
is  not  conducive  to  good  work  and  satisfactory  results. 

The  M.  A.  Seed  Dry  Plate  Company  make  two  brands  of 
orthochromatic  plates,  one  of  which  is  very  rapid. 

The  G.  Cramer  Dry  Plate  Company  have  slow,  medium,  and 
instantaneous  isochromatic  plates,  which  are  highly  esteemed 
by  some  microscopists  for  photo-micrographic  work. 

The  Stanley,  Hammer,  New  York,  and  Eastman  plates  are 
some  other  brands  which  receive  commendation  from  photog- 
raphers, but  it  is  not  the  fortune  of  the  author  to  be  familiar  with 
these  plates,  and  they  are  simply  mentioned  because  commended 
by  workers  of  known  ability  in  microscopical  science. 

The  celluloid  films  put  up  in  daylight  rolls  by  the  Kodak  Com- 
pany have  already  been  mentioned  on  a  previous  page,  and  the 
student  is  referred  to  what  was  said  concerning  these  films  in 
that  portion  of  this  book. 

The  orthochromatic  plates  are  the  best  for  all  photo-micro- 
graphic  work,  because  they  give  color  values,  so  important  in 
photographing  stained  sections.  Without  these  plates  it  would 
be  impossible  to  secure  clear,  sharp  pictures  of  the  colored  sec- 
tions, because  the  ordinary  dry  plates  have  not  the  proper  tone 
values,  some  of  the  colors  appearing  too  dense,  while  others 


MAKING  THE   PHOTO-MICROGRAPHS  159 

are  too  light.  The  plate  is  orthochromatized  by  immersing  it  in 
a  solution  of  a  dye  belonging  to  the  eosin  group,  in  accordance 
with  the  theory  of  Professor  Draper,  who  discovered  that  only 
those  rays  which  are  absorbed  by  the  film  on  the  plate  produce 
chemical  changes  in  the  sensitive  surface.  In  using  these  ortho- 
chromatic  plates  it  is  necessary  to  interpose  between  the  lens 
and  the  surface  of  the  plate  a  color  screen  in  order  to  intercept 
and  equalize  the  chemical  action  of  the  different  colored  rays 
which  are  emitted  from  the  section.  The  color  of  the  screen  is 
generally  yellow,  although  it  is  claimed  by  some  experimenters 
that  a  tinge  of  red,  blue,  or  green  is  sometimes  preferred.  In 
the  use  of  these  plates  two  facts  must  be  borne  in  mind :  The 
exposure  must  be  prolonged  beyond  that  period  given  to  the 
ordinary  plates,  and  the  developing-tray  must  be  covered  while 
developing,  so  that  the  plates  will  not  be  fogged  by  the  light  sift- 
ing through  the  ruby  glass.  In  order  to  have  the  dark-room 
window  perfectly  safe,  it  will  be  well  to  cover  the  ruby  glass  with 
two  thicknesses  of  yellow  paper  or  cloth  specially  prepared  for 
this  purpose. 

Photo-micrographic   apparatus.  —  This  apparatus  is  generally 
united  into  one  series  and  consists  of  the  following  pieces :  — 

1.  A  compound  microscope. 

2.  A  camera  with  a  long  bellows. 

3.  A  massive  cast-iron  sole-plate. 

4.  Optical  bench  upon  which  the  accessories  and  auxiliaries 
are  placed  in  a  line  one  behind  the  other  ;  the  accessories  consist- 
ing of  — 

5.  Absorbing  cell  to  extract  heat  rays. 

6.  Condensers  for  bringing  the  light  to  focus. 

7.  Iris  diaphragm. 

8.  Ray  filter  containing  the  coloring  solution  for  photograph- 
ing colored  objects  with  orthochromatic  plates. 

9.  The  light,  either  oil,  ordinary  gas,  acetylene,  electric,  or 
sunlight. 

Several  firms  have  given  to  the  microscopist  some  excellent 
combinations  of  the  above  apparatus,  so  that  photo-micrographic 


1 60 


BIOLOGICAL  LABORATORY   METHODS 


work  is  now  accomplished  with  the  least  degree  of  inconvenience. 
It  simply  becomes  a  question  of  how  much  money  the  laboratory 
is  able  to  invest  in  this  important  adjunct  to  the  outfit. 

One  of  the  most  useful  simple  photo-micrographic  outfits  on 
the  market  is  the  one  illustrated  in  Figs.  86  and  87.     It  is  made 


FIG.  86.  —  Combined  Horizontal  and  Vertical  Photo-micrographic  Camera. 

(Zeiss.) 

by  the  Zeiss  Optical  Company  and  is  constructed  for  working 
in  either  a  vertical  or  horizontal  position.  The  illustrations  give 
a  very  clear  conception  of  this  instrument.  Any  first-class  com- 
pound microscope  can  be  adapted  to  this  outfit.  The  light  is 
obtained  from  the  gas-mains  with  the  use  of  the  Welsbach 


1 62        BIOLOGICAL  LABORATORY  METHODS 

mantle.  In  using  this  instrument,  or  any  other  of  the  same 
kind,  care  must  be  taken  to  see  that  the  connections  between 
the  camera  and  the  microscope  are  securely  made,  so  that  no 
white  light  can  possibly  enter  the  apparatus  except  that  which 
comes  through  the  objective  at  the  proper  time. 

When  the  income  of  the  laboratory  will  permit  of  a  larger  ap- 
propriation to  this  department  than  is  required  for  the  purchase 
of  the  last-described  photo-micrographic  outfit,  the  author  would 
advise  the  addition  of  the  larger  combination  like  that  shown  in 
the  illustration  (Fig.  88),  or  the  equally  valuable  one  made  by  the 
Bausch  &  Lomb  Optical  Company.  The  one  given  in  the  illus- 
tration is  made  by  the  Zeiss  Optical  Company  of  Germany, 
and  is  complicated,  but  very  complete  in  the  efficient  working 
of  all  its  parts.  The  laboratory  having  such  an  outfit  in  its 
equipment  is  indeed  fortunate,  for  there  seems  to  be  nothing 
wanting  to  enable  the  operator  to  produce  photo-micrographs  of 
the  highest  degree  of  perfection. 

The  essential  parts  of  the  outfit  are :  — 

1.  An  adjustable  sole-plate,  on  which  rests  the  microscope. 
Underneath  this  is  an  elevating-gear  for  raising  or  lowering  the 
sole-plate  when  microscope-stands  of  varying  heights  are  used. 

2.  A  horizontal   prismatic  steel  rail,  on  which  is  arranged 
axially  the  parts  of  the  apparatus  for  controlling  the  action  of 
the  light.     This  rail  is  called  the  "  optical  bench."     See  sr, 
page  163. 

3.  On  this  optical  bench  are  the  following  parts:  A  double 
lens  S,  for  the  object  of  rendering  the  rays  of  light  parallel ;  a 
single  front  lens  S',  which  serves  to  bring  the  rays  to  a  focus ; 
a  water  chamber  A,  containing  water  interposed  between  the 
.two  lenses  in  order  to  absorb  the  heat  rays ;  a  ray  filter  C,  for 
use  with  orthochromatic  plates  and  for  color  values ;  iris  dia- 
phragm /,  containing  spring-clips  for  holding  ground  glass  to 
use  in  centring  the  light,  for  colored  screen,  and  such  other 
purposes. 

The  optical  bench  and  the  sole-plate  with  the  microscope, 
Mk>  are  located  on  a  firm  iron  table  with  a  wooden  top,  which 


I 

o 

a 

i 


1 64        BIOLOGICAL  LABORATORY  METHODS 

gives  great  solidity  and  prevents  vibration  when  properly  located. 
On  one  end  of  this  table  is  also  placed  the  lamp,  EL  The  ap- 
paratus from  the  lamp  to  the  microscope  is  covered  with  a  board, 
Bd-Bd],  and  on  each  edge  of  this  board  is  suspended  a  dark 
cloth  for  the  purpose  of  excluding  all  side  light  after  the  appara- 
tus is  adjusted  and  the  plate  is  in  position  ready  to  be  exposed. 
On  the  second  table  TT  are  the  camera  and  the  adjusting 
rod  Ad,  which  is  used  to  make  sharp  focussing  when  the  operator 
is  sitting  at  the  ground  glass  Gr.  The  camera  bellows  is 
150  cm.  long  and  is  made  in  two  parts,  so  that  the  camera  may 
be  shortened  one-half  when  desired,  and  a  second  glass  plate  at 
Gr'  is  used  for  focussing  the  image  of  the  object,  when  a  less 
magnification  is  required. 

4.  Lantern-slide  carrier,  with  micrometer  movement. 

5.  Movable  dark  slide  for  determining  the  sensitiveness  of 
the  plate  with  the  different  powers  of  objectives.     The  plate  is 
moved  through  the  slide  in  such  a  way  as  to  expose  only  a  small 
strip  at  a  time,  so  that  it  is  necessary  to  use  only  one  plate  to 
determine  the  ratios. 

The  Bausch  &  Lomb  Optical  Company's  photo-micrographical 
outfit  is  built  somewhat  on  the  same  general  plan  as  that  just 
described;  there  are  some  advantages  in  one  that  are  not  to 
be  found  in  the  other ;  for  instance,  in  a  recent  improvement  in 
the  Bausch  &  Lomb  apparatus  the  table  carrying  the  optical 
bench  and  microscope  is  made  with  only  one  leg  and  tripod 
base,  and  the  top  is  fixed  so  that  it  will  revolve  horizontally. 
This  enables  the  operator  to  turn  the.  table-top  to  one  side  and 
focus  the  microscope  before  projecting  the  image  on  the  ground 
glass.  This  is  a  considerable  advantage  over  the  Zeiss  pattern, 
because  it  enables  the  operator  to  avoid  the  uncomfortable 
position  required  in  adjusting  the  microscope. 


CHAPTER   XI 

PHOTO-MICROGRAPHS  (CONTINUED)  —  SUNLIGHT  AND  ELEC- 
TRIC LIGHT,  OXY-HYDROGEN,  ACETYLENE,  CITY  GAS,  AND 
OIL  LAMPS,  AND  HOW  TO  USE  THEM 

WITH  the  great  improvements  developed  in  electricity  within 
the  past  few  years,  the  microscopist  has  but  little  room  for  com- 
plaint in  regard  to  the  lighting  of  the  apparatus  for  photograph- 
ing his  mounts.  In  those  places,  therefore,  where  electricity  can 
be  secured  no  other  kind  of  illumination  should  be  thought  of  in 
photo-micrography.  But  it  is  not  in  the  power  of  every  student 
to  use  this  most  excellent  light,  and  he  is  forced  to  look  in  other 
directions  for  illumination.  Sunlight  is,  of  course,  preferable, 
but  there  are  two  difficulties  in  the  way  of  its  use :  (i)  Its  in- 
tensity is  a  very  uncertain  quantity  in  doing  delicate  work,  and 
it  is  exceedingly  difficult  to  determine  the  exact  time  of  exposure 
in  each  instance  while  the  sun  is  gradually  climbing  the  zenith, 
and  the  rays  are  becoming  constantly  more  and  more  powerful 
in  their  effects  on  the  sensitive  plate.  (2)  The  sunlight  is  not 
always  available,  because  of  the  occurrence  of  cloudy  weather. 

When  it  is  possible  to  use  the  sunlight,  and  the  operator  pre- 
fers to  do  so,  there  must  be  attached  to  the  apparatus  a  heliostat 
and  porte-lumiere,  or  similar  contrivance,  for  controlling  the 
direction  of  the  rays  from  the  sun,  and  concentrating  them  at 
the  right  point  for  illuminating  the  object.  The  heliostat  is  an 
instrument  with  clockwork  construction,  which  causes  the  reflect- 
ing mirror  to  follow  the  sun,  and  thus  project  the  rays  into  the 
tube  of  the  microscope,  requiring  but  little  if  any  adjustment  of 
screws  on  the  part  of  the  manipulator. 

Electricity.  —  Although  this  source  of  light  is  not  as  bright  as 
direct  sunlight,  still  the  author  considers  it  to  be  preferable,  for 

165 


1 66  BIOLOGICAL   LABORATORY  METHODS 

the  reason  that  it  is  possible  to  have  it  under  thorough  control. 
Moreover,  the  intensity  is  entirely  sufficient  for  .all  purposes  in 
the  laboratory,  and  the  use  of  sunlight  for  projection  may  be 
dispensed  with. 

Electricity  is  used  in  the  production  of  two  kinds  of  light,  the 
incandescent  and  the  arc.  The  latter  is  the  most  intense,  but 
for  a  long  time  it  was  not  practicable  to  use  it  for  projection,  for 
the  reason  that  the  carbons  were  so  irregularly  consumed  it  was 
impossible  to  keep  the  arc  in  the  same  position  in  the  optical 
centre  of  the  condensers.  Not  until  within  very  recent  time 
has  this  difficulty  been  overcome.  To  Messrs.  J.  B.  Colt  & 
Company  is  much  of  the  credit  due  for  devising  an  arc  lamp 
which  seems  to  satisfy  all  the  demands  reasonably  made  of  such 
a  light.  The  Scientific  American,  in  speaking  of  this  most  excel- 
lent invention,  says  : l  — 

"  The  lamp  is  perfectly  automatic  in  the  control  of  the  carbons, 
which  are  disposed  at  such  an  angle  as  to  present  the  crater  of 
the  positive  carbon  to  the  condensing  lenses  of  the  projector. 
This  feature  is  very  desirable,  for,  when  carbons  are  vertical, 
even  if  the  negative  carbon  is  advanced  toward  the  condenser 
out  of  line  of  the  positive,  the  light  will  proceed  from  the  nega- 
tive carbon  as  well  as  the  positive,  thus  making  two  sources  of 
light  instead  of  one  —  a  condition  fatal  to  definition  on  high- 
class  work.  If,  however,  the  carbons  are  placed  at  an  angle,  as 
shown  in  the  cut,  the  luminous  spot  on  the  negative  carbon  is 
obscured  from  the  condenser,  and  the  crater  on  the  positive 
carbon  is  presented  in  a  most  favorable  way.  By  an  admirable 
system  of  mechanism  in  the  lamp  referred  to,  the  point  of  the 
lower  carbon  is  accurately  maintained  in  a  given  position,  and 
the  upper  carbon  is  gradually  and  regularly  fed  toward  it  in  the 
exact  proportion  to  its  consumption.  This  mechanism  is  so 
simply  and  nicely  adjusted  that  the  lamp  may  be  run  for  hours 
without  a  flicker,  always  maintaining  the  radiant  in  the  optical 
centre  of  the  projector. 

"  The  regulating  device  is  contained  in  a  metal  case,  only  2 

1  Scientific  American,  January  20,  1894. 


PHOTO-MICROGRAPHS  —  ELECTRICITY 

inches  thick  by  3}  inches  wide  and  4^  inches  high,  making  it 
the  most  compact  and  easily  adjusted  lamp  that  has  been  brought 
to  our  notice. 

"  The  negative  carbon  is  automatically  moved  upward  as  it 
burns  away.  The  positive  carbon  is  fed  down  by  the  weight  of 
the  carbon  holder  and  by  a  small  spring-actuated  train  of  gear- 
ing, which  is  held  in  check  while  the  arc  is  of  normal  length,  or 
released  when  the  length  of  the  arc  becomes  too  great,  by  a 
shunt  magnet  contained  by  the  casing.  The  action  of  the  shunt 
magnet  is  controlled  by  a  spring  acting  on  its  armature  ;  an  in- 
crease in  the  tension  of  the  spring  increases  the  length  of  the 
arc,  while  a  reduction  of  the  tension  diminishes  the  length  of 
the  arc. 

"  The  mechanism  controlling  the  carbons  is  so  constructed 
that  the  lamp  may  be  used  on  from  five  to  twenty  amperes  of 
current  by  the  mere  inserting  of  carbons  of  suitable  sizes.  The 
lamp  is  perfectly  insulated,  so  it  may  be  freely  handled. 

"  This  lamp  may  be  clamped  on  a  vertical  post,  with  a  sliding 
base,  admitting  of  universal  adjustment,  or  it  may  be  mounted 
otherwise  as  may  be  desired.  The  utility  of  this  lamp  is  by  no 
means  confined  to  optical  projection,  for,  owing  to  its  convenient 
size  and  the  absolute  steadiness  of  its  light,  it  finds  extensive 
use  in  photo-lithography  and  copying,  micro-photography  for 
theatrical  effects,  and,  in  fact,  all  uses  for  which  an  intense 
artificial  light  is  desired.  The  intensity  of  this  light  admits  of 
the  use  of  the  optical  projector  in  a  room  that  is  only  partially 
darkened,  which  greatly  increases  its  utility  and  widens  its 
scope." 

In  Fig.  89,  which  is  a  representation  of  this  lamp,  A  is  a  screw 
for  raising  or  lowering  the  entire  apparatus,  so  that  proper  cen- 
tring of  the  light  may  be  accomplished  ;  B  is  a  knob  used  in 
separating  the  carbons  when  the  consumption  requires  the  sub- 
stitution of  others  ;  the  magnets  shown  at  D  are  for  the  purpose 
of  separating  the  carbons  when  the  current  is  turned  on  ;  E  is 
the  adjustment  to  enable  the  operator  to  place  the  lower  pencil 
in  the  best  position ;  C  is  a  screw  for  adjusting  the  apparatus 


1 68  BIOLOGICAL  LABORATORY   METHODS 

laterally,  while  on  the  opposite  side  is  a  spring  to  hold  the 
instrument  firmly  against  C. 

As  in  the  case  of  other  electrical  appliances,  a  rheostat  is  re- 
quired in  the  use  of  this  lamp,  and  the  one  illustrated  in  Fig.  90 
is  suitable  for  low-voltage  currents.  This  rheostat  is  made 
by  the  same  firm  who  manufacture  the  electric  lamp  shown  in 
Fig.  92. 

Some  suitable  form  of  storage  battery  will  be  found  convenient 
where  a  very  steady  light  is  demanded,  although  the  lamp  above 


FIG.  89.  —  Colt's  Electric  Lamp. 

illustrated  will  give  a  most  constant  flame  from  currents  obtained 
directly  from  the  dynamos.  About  thirty  cells  of  the  storage 
battery  will  be  required  to  run  the  lamp.  The  current  which 
comes  either  from  the  dynamo  or  the  secondary  battery  must  be 
direct  and  not  alternating,  and  it  should  be  from  five  to  fifteen 
amperes  and  not  less  than  sixty  volts  in  pressure.  A  safety 
fuse  should  be  placed  in  the  circuit,  so  that  if  any  accident 
occurs,  by  short-circuiting,  the  installation  will  not  be  injured. 
If  a  short-circuit  takes  place  while  the  wires  are  being  attached 
to  the  lamp,  the  safety  fuse  will  be  immediately  burnt  out  before 
the  instrument  is  affected.  Inasmuch  as  the  electromotive 
force  of  the  current  usually  received  directly  from  the  dynamos 


PHOTO-MICROGRAPHS  —  ELECTRICITY 


I69 


is  1 10  volts,  it  is  necessary  to  introduce  the  rheostat  or  resist- 
ance coils  (see  Fig.  90)  in  order  to  give  the  correct  voltage  at 
the  lamp  terminals. 

Before  the  lamp  is  lighted  the  two  carbons  are  in  contact,  and 
as  soon  as  the  current  begins  to  flow  the  ends  of  the  carbons 
become  heated,  and  the  mechanism  of 
the  lamp  separates  them  immediately, 
and  the  arc  is  at  once  established. 
The  theory  explaining  this  action  is 
that  electricity,  at  the  usual  pressure, 
will  not  pass  through  a  space  unless 
filled  with  the  particles  of  the  incan- 
descent carbon.  When  the  separation 
takes  place,  the  arc  is  maintained  by 
the  vapor  or  particles  of  the  highly 
heated  carbon.  In  connecting  up  the 
wires  to  the  lamp  it  must  be  remem- 
bered that  the  carbon  containing  the 
crater  must  be  placed  in  connection 
with  the  positive  pole  of  the  dynamo, 
or  battery,  and  the  carbon  containing 
the  point  must  be  joined  to  the 
negative  pole.  The  first,  or  positive,  pencil  must  be  at  the 
top  of  the  lamp,  and  both  pencils  must  be  inclined,  so  that  the 
light  will  not  be  projected  downward,  as  will  be  the  case  if 
the  carbons  are  kept  in  a  vertical  position,  but  thrown  out  directly 
in  front  along  the  optic  axis.  There  is  a  limit,  however;  to  this 
inclination,  because,  since  the  lower  pencil  is  slightly  in  front  of 
the  upper,  too  much  inclination  will  cause  the  point  of  the  lower 
carbon  to  be  projected  on  the  screen  in  the  form  of  a  shadow. 
The  best  length  to  be  given  to  the  arc  for  steady  results  is  from 
-J-  to  T8g-  of  an  inch.  When  it  is  too  short,  there  is  an  unpleasant 
hissing  and  spluttering  noise.  In  the  lamp  illustrated  in 
Fig.  89,  there  is  a  mechanism  provided  for  controlling  the  dis- 
tance between  the  carbons.  The  positive  carbon  is  consumed 
faster  than  the  negative,  and  it  is  at  all  times  kept  in  the  form 


FIG.  90.  —  Rheostat. 


I/O  BIOLOGICAL  LABORATORY  METHODS 

of  a  crater,  from  the  depths  of  which  come  the  luminous  rays 
that  are  projected  into  the  lantern. 

Oxy-hydrogen,  or  lime  light.  —  This  light  is  produced  by  the 
heating  to  incandescence  a  stick  of  lime  under  the  combined 
action  of  burning  oxygen  and  hydrogen  gases.  But  little  need 
be  said,  however,  concerning  this  method  of  lighting,  since  the 
electric  current  is  so  readily  secured  in  any  well-equipped  labora- 
tory, and  there  is  so  little  inconvenience  and  danger  connected 
with  its  use.  The  oxy-hydrogen  illumination,  unless  managed 
with  the  greatest  care,  is  liable  to  give  serious  trouble,  and 
there  is  always  an  element  of  danger  associated  with  it,  because 
of  the  explosive  properties  of  the  gases,  which  make  its  use  in 
the  hands  of  students  unsafe.  If,  however,  such  a  light  is 
desired,  the  gases  can  be  purchased  compressed  in  metal 
cylinders  ready  for  immediate  use  with  but  little  cost.  Or,  these 
gases  can  be  generated  in  the  laboratory  with  but  little  trouble, 
provided  the  apparatus  is  secured.  The  oxygen  is  made  by 
heating  in  a  retort  a  mixture  of  one  part  of  black  oxide  of  man- 
ganese with  four  parts  of  chlorate  of  potash.  Be  careful  to  have 
the  retort  perfectly  clean  and  thoroughly  dry,  and  use  only  the 
purest  chemicals.  If  they  are  pure,  they  will  melt  on  an  iron 
spoon  over  a  lamp  without  an  explosion.  Pass  the  gas  from  a 
retort  through  a  purifying-bottle  and  then  into  the  metal  cylinder. 
See  that  all  connections  are  open  and  clear  of  all  obstructions, 
and  let  the  gas  flow  a  short  time  before  connection  with  the 
cylinder.  All  the  air  possible  must  be  excluded  from  all  parts 
of  the  apparatus  to  insure  good  results. 

The  hydrogen  gas  is  generated  by  the  action  of  sulphuric  acid 
on  metal  zinc.  Pass  the  gas  through  a  purifying  bottle,  as 
in  the  case  of  the  oxygen,  and  the  same  care  must  be  taken 
in  its  preparation  as  indicated  in  the  manufacture  of  oxygen,  in 
order  to  prevent  explosions  and  serious  damage  to  person  and 
property. 

The  following  apparatus  will  complete  the  outfit  for  producing 
the  calcium  light :  — 

Oxygen  and  hydrogen  cylinders. 


PHOTO-MICROGRAPHS  —  GAS   LAMP 


171 


Pressure  gauges  for  the  cylinders  to  measure  their  contents 
(see  Fig.  91). 

Light-regulator  valves. 

Oxy-hydrogen  mixing  jet  for  holding  the  lime  pencil  and  mix- 
ing the  gases  before  they  strike  the  lime. 

Hood  for  holding 
the  lamp  fixtures. 

And,  if  the  gases 
are  made  in  the  lab- 
oratory, generators, 
retorts,  wash  bot- 
tles, and  rubber 
tubing. 

Gas  Lamp.  —  The 
best  form  is  Wels- 
bach's  gas  burner 
and  reflector  as  de- 
vised by  J.  B.  Colt 
&  Company.  This 
burner  gives  a 
strong  light,  and  will 
answer  very  well 
where  electricity  or 
the  lime  light  is 
not  available.  In 
fact,  it  will  serve 
most  excellently  in  all  cases  where  low-power  objectives  are  used. 

Acetylene-gas  light.  —  There  are  several  different  kinds  of 
lamps  and  generators  on  the  market  for  burning  and  manufactur- 
ing acetylene  gas,  but  all  the  generators  are  constructed  upon  the 
principle  of  immersing  calcium  carbide  in  water.  Calcium  car- 
bide is  a  crystallization  caused  by  fusing  with  an  intense  electric 
energy  a  combination  of  powdered  coke  and  lime  in  proper  pro- 
portions. The  gas  is  generated  by  bringing  the  calcium  carbide 
in  contact  with  water,  when  immediately  a  colorless  gas  is  gen- 
erated which  is  unaffected  by  variations  of  temperature.  The 


BIOLOGICAL   LABORATORY   METHODS 


light  given  by  this  gas  is  very  bright  and  pure  in  color,  and  is 
very  much  like  the  light  received  from  the  sun.  The  carbide  is 
very  cheap,  costing  less  than  3!  cents  per  pound,  and  the  in- 
creased use  of  the  gas  will  make  it  still  cheaper.  '  One  pound  of 
the  carbide  will  develop  about  five  cubic  feet  of  gas. 

Calcium  carbide,  when  in  the  solid  form  and  properly  stored 
and  protected  from  contact  with  water,  is  non-explosive.  The 
greatest  precaution,  however,  must  be  used  in  the  manufacture  of 
the  gas  because  of  its  explosive  properties,  but  it  is  perfectly 

safe,  if  due  care  is  taken  in  placing 
the  carbide  in  the  generators  to  see 
that  fire  is  not  brought  in  range  of  the 
apparatus,  and  that  there  are  no  leaks 
in  the  pipes  conducting  the  gas  to  the 
burners. 

The  burner  has  a  peculiar  shape, 
differing  from  that  given  to  the  burn- 
ers used  with  the  ordinary  city  gas. 
The  illustration  shown  in  Fig.  92  rep- 
resents the  usual  form  adopted  for 
these  burners.  This  is  the  one  used 
by  J.  B.  Colt  &  Company  with  their 
acetylene  generators. 
Oil  light.  —  Sometimes  it  is  impossible  to  obtain  either  the 
electric,  the  oxy-hydrogen,  the  acetylene,  or  the  gas  light,  and 
under  these  circumstances  a  good  oil  lamp  will  render  efficient 
service,  if  the  light  is  of  that  nature  furnished  by  a  well-con- 
ditioned student's  kerosene-oil  lamp.  Of  course  it  must  be 
remembered  that  the  light  from  such  a  source  is  by  no  means 
as  brilliant  as  that  secured  from  either  of  the  lamps  already 
described,  and  that  the  exposure  will  be  correspondingly  long 
in  making  the  photo-micrographs  with  kerosene-oil  light. 

The  author  has  seen  some  beautiful  photo-micrographs  made 
by  the  oil  light  with  the  high  powers  of  objectives,  but  he  well 
remembers  that  the  work  was  done  by  the  hands  of  skilful 
operators. 


FIG.  92.  — Colt's  Acetylene 
Gas  Burner. 


CHAPTER   XII 

PHOTO-MICROGRAPHS    (CONTINUED) PHOTOGRAPHIC    DRY 

PLATES    AND    MAKING    THE    NEGATIVES 

THE  student  should  begin  with  the  low-power  objectives  first 
and  not  attempt  too  difficult  work  until  he  has  mastered  the 
simplest  details.  The  arrangement  of  the  apparatus  must  be 
carefully  looked  after  and  the  parts  all  accurately  adjusted  so 
that  all  of  the  glasses  will  occupy  positions  in  the  same  optic 
axis.  There  must  not  be  the  slightest  leak  of  light  in  any  por- 
tion of  the  outfit.  It  is  best  to  use  with  the  objective  the 
projection  ocular  described  on  page  35,  although  good  work 
has  been  done  in  the  past  by  some  of  our  leading  microscopists 
by  the  use  of  the  objective  alone.  Only  the  best  glasses  must 
be  used  in  this  work,  because  it  will  be  found  very  difficult  to 
make  the  actinic  and  visual  foci  coincide.  With  the  use  of  the 
Jena  apochromatic  lenses,  however,  this  difficulty  is  almost 
entirely  overcome,  and  it  is  possible  to  secure  objectives  which 
are  specially  made  for  use  with  the  photo-micrographic  appa- 
ratus, with  which  when  the  visual  image  is  focussed  on  the 
ground  glass  an  image  equally  sharp  in  its  actinic  effects  is 
projected  on  the  sensitive  surface  of  the  dry  plate. 

Mr.  T.  Comber,  in  the  Journal  of  the  Liverpool  Microscopical 
Society,  gives  the  following  steps  for  adjusting  the  photo-micro- 
graphic  apparatus  with  sunlight  as  the  illuminant :  — 

"  i.  Accurately  centre  the  achromatic  condenser,  regulating 
the  diaphragm  so  that  the  opening  is  a  little  smaller  than  the 
field. 

"  2.  Remove  the  mirror  from  the  heliostat,  to  ascertain  that 
the  spindle  appears  precisely  on  and  in  the  field,  which  it 
should  do  if  the  heliostat  has  been  properly  placed.  Exactness 


BIOLOGICAL   LABORATORY   METHODS 

in  this  adjustment  is  necessary,  otherwise  the  beam  of  sunlight 
will  not  be  motionless.  The  mirror  is  then  replaced  to  reflect 
the  sun  in  the  centre  of  the  field.  At  this  stage  the  eye  must  be 
protected  by  a  dark-colored  glass  placed  below  the  condenser. 

"  3.  The  object  being  placed  on  the  stage  is  brought  into  the 
centre  of  the  field  and  focussed. 

"  4.  The  condenser  is  next  focussed  to  throw  the  sun's  image 
exactly  in  the  plane  of  the  object.  Sharpness  of  the  ultimate 
image  upon  the  ground  glass  cannot  be  secured  without  this. 

"5.  Changing  the  objective  to  one-sixth  (4  mm.),  I  next 
measure  the  thickness  of  the  cover-glass,  or  rather  the  distance 
between  that  plane  of  the  object  which  it  is  desired  to  photo- 
graph and  the  upper  surface  of  the  cover-glass,  by  means  of  the 
fine  adjustment  screw.  The  purpose  of  this  is  twofold  :  ist,  To 
facilitate  cover  correction ;  2d,  To  ascertain  whether  the  2  mm. 
object-glass,  which  is  now  put  on,  can  get  down  to  it,  for  its 
front  lens  is  rather  more  than  a  hemisphere.  .  .  . 

"6.  The  illuminating  cone  thrown  by  the  condenser  has  to 
be  regulated.  The  width  of  the  cone  should  vary  according 
to  the  nature  of  the  object  and  the  quality  of  the  object-glass. 
Too  narrow  a  cone  produces  diffraction  fringes,  that  bane  of 
photo-micrography;  too  wide  a  cone  produces  haze.  To  get 
true  images,  the  cone,  whether  it  be  wide  or  narrow,  must  be 
absolutely  axial.  Even  a  very  slight  obliquity  renders  the 
images  unreliable. 

"  7.  The  ordinary  eyepiece  is  now  changed  for  a  projection 
eyepiece,  set  at  the  distance  at  which  the  sensitive  plate  is  to 
stand;  the  camera  is  attached  and  the  long  focussing  rod 
coupled  on  (see  Fig.  88).  The  image  of  the  sun  will  be  found 
in  the  centre  of  the  ground  glass.  If  it  is  not,  the  centring  of 
the  condenser  must  be  wrong,  and  will  require  alteration.  The 
sun's  image  should  be  sharp  at  the  edge,  unless  the  sky  is  hazy. 
The  image  of  the  object,  as  seen  against  that  of  the  sun,  will  be 
somewhat  out  of  focus,  but  a  slight  turn  of  the  focussing  rod 
brings  ft  right."1 

1  See  also  Jour.  Roy.  Mic.  Soc. 


PHOTO-MICROGRAPHS  175 

The  instructions  contained  in  the  above  copy  from  the  article 
by  Mr.  Comber  will  also  apply  when  the  electric  lamp  is  used 
in  place  of  the  sun.  Proceed  in  the  same  manner  in  all  respects 
while  focussing,  except  that  the  electric  lamp  is  substituted  for 
the  heliostat. 

Since  it  is  the  common  practice  of  microscopists  to  stain  the 
sections,  in  order  that  distinctive  contrast  in  the  systems  of 
cellular  structure  may  be  secured,  it  will  be  found  difficult  to 
produce  good  negatives,  presenting  clearness  in  detail,  with  the 
use  of  the  ordinary  dry  plate.  It  has  been  found  necessary, 
therefore,  to  submit  the  plates  to  a  special  treatment,  so  as  to 
correct  them  for  color  values.  The  student  can  prepare  the 
ordinary  dry  plates  by  immersing  them  in  erythrosin  and  cyanin 
solutions.  When  so  treated  the  plates  are  called  orthochromatic, 
and  they  may  be  purchased  from  dealers,  sensitized  for  all  the 
colors  of  the  spectrum.  A  most  excellent  account  of  the  method 
for  preparing  the  plate  is  given  in  Anthony's  Photographic  Bul- 
letin for  -ffipj,  p.  608,  extracts  from  which  are  given  below. 
The  article  from  which  these  items  were  gathered  was  read 
before  the  Societ£  Francaise  de  Photographic,  on  May  5,  by 
M.  Monpillard.1 

"Orthochromatic  sensitiveness.  —  Though  chemical  orthochro- 
matic plates  are  sensitive  for  the  green  and  red,  and  generally 
give  satisfaction,  M.  Monpillard  says  that,  for  scientific  pur- 
poses, he  prefers  ready  orthochromatized  plates,  which,  when 
used  shortly  after  preparation,  have  a  maximum  of  sensitiveness 
to  the  luminous  radiations.  The  operation  of  orthochromatiza- 
tion  demands  only  elementary  care.  The  dark-room  lamp  should 
have  two  thicknesses  of  deep  ruby  glass,  the  flame  being  re- 
duced to  as  small  a  degree  as  convenient  during  the  bathing  of 
the  plates.  After  the  plates  are  bathed  they  are  passed  through 
three  dishes  of  distilled  water,  and  are  finally  dried  in  a  drying 
cupboard  containing  a  vessel  in  which  calcium  chloride  is 
placed. 

"  For  photo-micrographic  purposes  the  following  colors  give 

1  See  also  Jour.  Roy,  Mic.  Soc.  for  February,  1894,  p.  113. 


176  BIOLOGICAL   LABORATORY  METHODS 

the  best  results :  (i)  Erythrosin  (for  green-yellow,  yellow,  and 
yellow-orange)  ;  (2)  Cyanin  (for  red-orange  and  red). 

"  M.  Monpillard  says  the  following  formulae  have  given  him 
satisfaction :  — 

"  Erythrosin  (stock  solution) :  Erythrosin,  i  part ;  distilled 
water,  1000  parts. 

"  Sensitizing  bath  :  Stock  solution  of  erythrosin,  4  cc. ;  water 
100  cc.  ;  ammonia,  0.5  cc. 

"Cyanin  (stock  solution):  Cyanin,  o.i  part;  alcohol  (95  per 
cent),  100  parts.  Only  a  small  quantity  of  the  solution  should 
be  prepared,  and  it  should  be  kept  in  the  dark. 

"  Sensitizing  bath :  Stock  solution  of  cyanin,  4  cc.  ;  water, 
100  cc. ;  alcohol  (95  per  cent),  5  cc.  ;  ammonia,  1.5  cc.  The 
plates  are  immersed  in  either  of  the  foregoing  baths  for  two 
minutes,  and  are  then  washed  and  dried  as  directed. 

"  Erythrosin  and  cyanin  plates  bathed  in  both  erythrosin  and 
cyanin  are  rendered  sensitive  to  yellow  and  red.  The  first  bath 
consists  of:  Stock  solution  of  erythrosin,  20  cc. ;  distilled  water, 
80  cc.  After  two  minutes'  immersion,  the  plates  are  washed  in 
two  waters,  and  are  then  bathed  in  the  cyanin  solution  given, 
washed  and  dried. 

"  Plates  so  treated  are,  it  is  pointed  out,  very  much  slower,, 
but  this  is  no  disadvantage  in  photo-micrography,  and  on  the 
other  hand  they  do  not  fog  in  development,  which  frequently 
happens  when,  to  raise  their  general  sensitiveness,  the  ortho- 
chromatizing  bath  is  preceded  by  an  alkalin  bath. 

"  Colored  screens.  —  Colored  screens  may  be  used  either  in 
the  form  of  stained  collodion,  or,  preferably,  a  small  glass  trough, 
-with  parallel  faces,  may  be  fitted  with  either  of  the  following 
solutions :  — 

"  (i)  For  light  yellow  screen :  Neutral  chromate  of  potash, 
i  grm.  ;  water,  100  parts. 

"  (2)  For  deep  yellow  screen  :  Neutral  chromate  of  potash, 
5  grms. ;  water,  100  parts. 

"  (3)  For  orange  screen :  Bichromate  of  potash,  8  grms. ; 
water,  100  parts. 


PHOTO-MICROGRAPHS  177 

"(4)  For  red  screen:  Erythrosin,  0.2  grm. ;  water,  100 
parts. 

"  Number  i  weakens  the  blues  and  yellows ;  No.  2  extin- 
guishes them ;  No.  3  cuts  off  the  blue ;  No.  4  accentuates  the 
action  of  the  red. 

"  With  these  colored  screens,  and  having  sensitized  the  plates 
for  given  colors,  it  will  be  easy  to  obtain,  in  their  true  values, 
reproductions  of  objects,  colored  or  uncolored.  It  is  necessary, 
however,  that  the  focus  and  the  exposure  should  be  made  in 
the  same  monochromatic  light,  corresponding  to  a  determined 
spectrum  color.  This  method  of  working  assures  the  perfect 
sharpness  of  the  image,  inasmuch  as  the  chemical  focus  is  cor- 
rected. 

"  Where  an  object  combines  red  and  yellow  colors,  ...  it 
would  be  better  to  sensitize  for  red  and  yellow,  and,  according 
to  the  intensity  of  the  former,  expose  with  a  deep  yellow,  or 
orange,  screen.  If  blues  and  violets  are  found  in  the  presence 
of  yellows,  oranges,  or  reds,  it  would  suffice  to  use  a  plate  sensi- 
tized for  the  least  actinic  color  (yellow  or  red),  and  as  the  plate 
is,  of  course,  sensitive  to  the  blues  and  violets,  a  yellow  screen, 
pale  or  deep,  could  be  used  according  as  the  more  actinic 
parts  of  the  object  are  more  or  less  colored.  For  develop- 
ment, the  author  recommends  hydroquinon  with  an  alkalin  car- 
bonate and  bromide,  and  the  use  of  a  feeble  light  in  the  dark 
room." 

The  color  screen  can  be  placed  in  the  front  of  the  camera,  in 
such  a  position  that  all  the  rays  coming  from  the  object  must 
pass  through  it  before  reaching  the  sensitive  dry  plate. 

Exposures.  —  There  are  so  many  elements  controlling  the  de- 
gree of  exposure  that  it  is  quite  difficult  to  put  down  any  fixed 
rules  to  guide  the  operator.  The  character  of  the  light,  the 
power  of  the  objective  used,  and  the  condition  of  the  object,  are 
all  to  be  well  understood  before  this  question  can  be  determined, 
and  these  factors  can  only  be  known  after  careful  experiment. 
As  a  general  guide  it  may  be  stated  that  with  orthochromatic 
plates  used  in  connection  with  the  color  screen  the  following 

N 


BIOLOGICAL   LABORATORY   METHODS 

figures  give  about  the  limits  of  exposures.  These  results  have 
been  determined  by  Mr.  W.  H.  Walmsley,  who  has  done  some 
excellent  work  in  photo-micrography.  He  used,  however,  a  coal 
oil  or  petroleum  lamp  to  supply  the  illumination,  and  in  the  sub- 
stitution of  the  sunlight  or  electric  light  the  time  will  be  consid- 
erably reduced. 

i-J-inch  objective,  3  to  45  seconds. 

j-inch  objective,  J  to  i^  minutes. 
T%  objective,  $  to  3  minutes. 

^  objective,  2  to  7  minutes. 
Y1^  objective,  5  to  10  minutes. 

The  sensitive  plate  having  been  carefully  dusted  and  placed 
in  the  plate-holder  —  which,  of  course,  must  be  done  in  the  dark 
room  where  no  ray  of  white  light  can  strike  its  surface  —  and 
the  focussing  of  all  parts  of  the  apparatus  having  been  thoroughly 
accomplished,  as  already  explained,  the  exposure  is  made  by 
attaching  the  plate-holder  to  the  camera  in  the  place  of  the 
ground  glass.  The  light  is  first  excluded  from  the  apparatus 
by  placing  an  opaque  card  with  a  black  surface  between  the 
objective  lens  and  the  stage,  or,  if  very  high  powers  are  used, 
immediately  beneath  the  stage.  The  slide  is  now  withdrawn 
from  the  holder,  and,  with  watch  in  the  left  hand,  the  card  is 
quickly  raised,  the  light  permitted  to  enter  the  instrument  and 
project  the  image  of  the  object  on  the  sensitive  surface  of  the 
plate.  After  the  exposure  is  completed  quickly  return  the  card 
to  its  place,  and  then  push  in  the  slide.  Now  unfasten  the 
holder  from  the  camera,  and  return  with  it  to  the  dark  room  for 
the  purpose  of  developing  the  picture. 

Before  opening  the  holder  to  take  out  the  plate  prepare  the 
following  developing  and  fixing  solutions.  As  a  matter  of  illus- 
tration we  will  take  Carbutt's  orthochromatic  plates  to  show  the 
method  of  treatment,  and  his  formulae  are  given,  therefore,  in 
these  instructions  ;  but  the  student  will  find  at  the  close  of  this 
volume  other  developers  if  he  desires  to  use  plates  made  by  dif- 
ferent manufacturers. 


PHOTO-MICROGRAPHS 


179 


Eikonogen  and  Hydrochinon  Developer 
A 

Distilled  water 600  cc. 

Sulphite  of  soda  crystals  ......  120  grammes 

Eikonogen 22  grammes 

Hydrochinon io|  grammes 

Water  to  make  up  to 960  cc. 

B 

Distilled  water          .         .         .         .    '     .         .         .  600  cc. 

Carbonate  of  potash 60  grammes 

Carbonate  soda  crystals     ......       60  grammes 

Water  to  make  up  to         .         .         .         .         .         .  960  cc. 


A 

B 

Water 

For  instantaneous  exposures 

30  cc. 

30  cc. 

120  CC. 

"    portraits        

30   - 

30   « 

I50    « 

"    landscapes  .         .         .     (  Sen.  20-27  \ 
Full  exposures          .1     "    16-20  / 

30    " 

I5    « 

90    « 

"    lantern  slides        .         .         . 

30    « 

25    " 

120    " 

Full  exposures           .... 

30   " 

25    « 

120    " 

Add  2  to  6  drops  restrainer  D  to  each  ounce  of  developer  for  lantern  slides 
and  full  exposures. 

NOTE.  —  More  of  A  will  increase  density,  more  of  B  will  increase  detail 
and  softness.  The  temperature  of  the  developer  should  not  vary  much  below 
i8.3°C.  nor  above  24°  C. 


Restrainer 
D 


Bromide  potassa 
Water 


14  grammes 
150  cc. 


Select  a  suitable  tray  for  the  size  of  the  plate,  take  the  plate 
from  the  holder,  and  place  it  sensitive  side  up  in  the  tray,  and 
pour  over  it  quickly  the  developing  solution.  Now  gently  rock 
the  tray  back  and  forth,  so  that  fresh  portions  of  the  liquid  will 


180  BIOLOGICAL   LABORATORY   METHODS 

strike  every  part  of  the  surface,  as  is  shown  in  Fig.  74.  If  the 
plate  has  been  properly  exposed,  the  image  will  begin  to  show 
within  fifteen  to  thirty  seconds;  but  if  it  has  been  exposed  too 
long  to  the  action  of  the  light,  overexposed,  as  it  is  called,  the 
image  flashes  up  very  soon  after  the  developer  strikes  the  sur- 
face, and  then  rapidly  sinks  into  the  film  and  blackens  all  over 
the  surface.  If  an  underexposure  has  been  made,  the  image  fails 
to  appear  until  after  a  minute  or  more  has  passed,  and  the  details 
are  flat  and  faint,  the  ground  of  the  resulting  negative  being  a 
sickly  grayish  tint.  In  the  case  of  a  very  much  overexposed  or 
underexposed  plate  it  is  best  to  try  again,  and  repeat  the  opera- 
tion until  the  correct  exposure  is  approximately  attained. 

After  the  details  of  the  picture  have  come  out  well,  and  the 
image  begins  to  sink  gradually  into  the  film,  the  develop- 
ing action  is  stopped  by  pouring  the  solution  back  into  the 
graduate,  and  washing  the  plate  under  the  water-tap.  It  must 
be  remembered  that  the  orthochromatic  plate  is  very  sensitive, 
and  exposure  for  a  time  in  any  light  which  is  safe  for  the  ordi- 
nary plates  will,  after  a  short  time,  cause  the  negative  to  fog. 
The  development  must,  therefore,  be  almost  entirely  accom- 
plished with  the  tray  covered.  The  plate  is  now  placed  in  the 
fixing-bath,  and  permitted  to  remain  under  the  action  of  the 
solution  until  all  the  soluble  salts  of  silver  are  dissolved  out  and 
no  white  cloudiness  is  perceptible  on  the  back  of  the  plate. 
The  following  is  the  method  of  preparing  the  fixing  solution :  — 

Carbutfs  Add  Fixing  and  Clearing  Bath 

Sulphuric  acid 4  cc. 

Hyposulphite  of  soda     .         .         .         .         .         .  480  grammes 

Sulphite  of  soda     . 60  grammes 

Chrome  alum 30  grammes 

Warm  water           .......  1920  cc. 

During  cold  weather  use  only  half  the  quantity  of  chrome  alum 
given  above. 

"  Dissolve  the  hyposulphite  of  soda  in  48  ounces  (1440  cc.) 
of  water,  the  sulphite  of  soda  in  6  ounces  (180  cc.)  of  water, 


PHOTO-MICROGRAPHS  1 8 1 

mix  the  sulphuric  acid  with  2  ounces  (60  cc.)  of  water,  and 
pour  slowly  into  the  sulphite  of  soda  solution,  and  add  to  the 
hyposulphite,  then  dissolve  the  chrome  alum  in  8  ounces  (240  cc.) 
of  water,  and  add  to  the  bulk  of  solution,  and  the  bath  is  ready. 
This  bath  will  not  discolor  until  after  long  usage,  and  both 
clears  up  the  shadows  of  the  negative  and  hardens  the  film  at 
the  same  time." 

When  the  negative  has  been  fully  treated  with  the  fixing  solu- 
tion, it  may  be  taken  out  into  white  light  without  any  further 
danger  of  injury.  Before  exposing  to  white  light  all  the  soluble 
silver  must  be  dissolved  by  the  fixing-bath,  which  is  indicated  by 
the  disappearance  of  the  white  cloud  seen  from  the  back  of  the 
negative.  In  a  fresh  fixing-bath  this  will  require  about  ten 
minutes.  Before  drying  it  is  thoroughly  washed  in  running 
water  for  at  least  a  half-hour  until  all  of  the  hyposulphite  of 
soda  is  eliminated  from  the  film.  This  washing  is  most  con- 
veniently done  in  the  washing  box  illustrated  in  Fig.  82.  The 
prolonged  washing  is  necessary  because  the  hypo  salts  hold  on 
to  the  film  with  such  tenacity,  and,  unless  entirely  extracted,  the 
negative  will  soon  become  yellow  and  lose  its  brilliancy  and  value. 

After  complete  washing,  the  plate  is  swabbed  with  a  wad  of 
wet  cotton  and  placed  on  edge  in  the  drying-rack  to  dry  spon- 
taneously (see  Fig.  84).  The  drying  will  take  place  much  sooner 
if  the  rack  is  situated  in  a  draught  of  cool  air.  Care  must  be 
exercised,  however,  to  prevent  heat  reaching  the  plate,  but  the 
drying  must  be  in  the  shade  and  in  cool  air,  otherwise  the  film 
will  melt  and.  the  negative  be  destroyed.  When  thoroughly 
dried,  the  surface  of  the  negative  is  coated  with  a  varnish  to  pro- 
tect it  from  the  effects  of  the  printing  processes  while  making 
the  positives.  A  permanent  number  is  then  made  on  one  corner 
of  the  plate,  and  a  corresponding  number  is  also  placed  on  the 
envelope  or  preserver,  which  contains  the  name  of  the  object, 
date,  and  such  other  data  as  maybe  desired  for  future  reference. 
If  the  negative  requires  intensification,  as  is  sometimes  the  case, 
the  following  method  is  recommended  by  Carbutt,  and  is  most 
excellent :  — 


1 82  BIOLOGICAL   LABORATORY   METHODS 

Carbutfs  Intensifying  Solution 

No.  i 

Bichloride  of  mercury 1 6  grammes 

Chloride  of  ammonia 1 6  grammes 

Distilled  water 600  cc. 

No.  2 

Chloride  of  ammonia     ......  1 6  grammes 

Water 600  cc. 

"  Flow  sufficient  of  No.  i  over  the  negative  to  cover  it,  and 
allow  either  partially  or  entirely  to  whiten  ;  the  longer  it  is  allowed 
to  act  the  more  intense  will  be  the  result ;  pour  off  into  the  sink, 
rinse,  and  flow  over  No.  2,  and  allow  to  act  one  minute;  wash 
off  and  pour  over  or  immerse  in  dilute  ammonia  water  (i  i  cc.  of 
strong  ammonia  in  240  cc.  of  water).  In  place  of  ammonia  a 
10  per  cent  solution  of  sulphite  of  soda  may  be  used  until 
changed  entirely  to  a  dark  brown  or  black.  Wash  thoroughly 
and  dry." 

If  the  celluloid  films  are  used  for  making  the  negatives  instead 
of  the  glass  plates,  the  method  of  procedure  is  the  same,  except 
that  when  the  last  washing  has  been  accomplished,  the  films  are 
soaked  for  five  minutes  in  the  following  solution  in  order  to  cause 
them  to  remain  flat  and  not  curl  up :  — 

Water 750  cc. 

Glycerin .         .  30  cc. 

It  often  becomes  necessary  to  touch  up  the  negative  in  order 
to  remove  from  it  any  defects  that  occur  at  times  in  the  film,  and 
this  must  be  done  before  the  final  finishing  varnish  is  applied. 
For  retouching,  either  graphite  pencils  or  some  opaque  paint 
such  as  Gihon's  is  used.  The  best  pencils  for  this  purpose  are 
L.  &  C.  Hardtmuth's  (Vienna)  "black  chalk  points."  Figure 
85  shows  a  good  retouching-frame  for  holding  the  negative  and 
directing  the  light  during  the  operation  of  pencilling  out  the 
defects. 


EQUIPMENT  OF   DARK   ROOM  183 

The  equipment  of  the  dark  room.  —  Besides  the  items  men- 
tioned on  page  12,  the  dark  room  should  be  provided  with 
developing  pans,  or  dishes,  or  developing  trays,  of  the  following 
sizes  :  — 

3!  x  \\  inches.  4f  X  sf  inches. 

4-i  x  ^f  inches.  5^  X  8^  inches. 

7X9  inches. 

These  trays  must  always  be  kept  in  convenient  reach  near  the 
bottles  containing  the  developing  solutions,  and  they  should  be 
used  for  no  other  purposes.  The  best  trays  are  made  out  of 
hard  rubber  (Fig.  79),  with  two  grooves  running  the  length  of  the 
tray,  upon  which  the  plate  can  rest,  and  thus  furnish  a  con- 
venient means  of  raising  the  negative  when  it  is  desired  to 
examine  the  progress  of  the  developing. 

On  another  shelf  of  the  dark  room,  sufficiently  removed  from 
the  developing  shelf,  or  table,  to  avoid  all  chance  of  mixing 
the  chemicals,  the  hard  rubber  fixing-baths  to  contain  the  hypo- 
sulphite of  soda  solutions  may  be  located  (Fig.  81).  The  follow- 
ing sizes  will  be  found  suitable  for  most  purposes  :  — 

3i  X  \\  inches.  4X5  inches. 

5     x  7    inches.  5x8  inches. 

6\  x  8|  inches. 

These  fixing-bath  vessels  are  formed  in  such  a  manner  as  to 
hold  the  plates  in  a  vertical  position,  and  the  grooves  will  permit 
one  dozen  negatives  to  be  fixed  at  the  same  time.  The  sediment 
which  may  accumulate  is  kept  from  contact  with  the  soft  film  by 
a  ridge  rising  above  the  bottom  of  the  vessel. 

Several  graduates,  or  measuring  glasses,  of  4-ounce  or  120  cc. 
capacity  must  also  be  in  the  dark  room  for  enabling  the  opera- 
tor to  secure  the  proper  proportions  of  the  developing  agents 
(Fig.  80). 

A  minim  glass  will  also  be  essential. 

The  light  used  in  the  dark  room  must  be  carefully  covered 
with  ruby  glass  and  also  two  thicknesses  of  yellow  paper,  or 


184  BIOLOGICAL  LABORATORY   METHODS 

glass,  to  insure  the  proper  protection  of  the  plates  while  trans- 
ferring them  to  the  plate-holder  and  developing.  If  electricity 
is  not  available,  a  gas  lamp  may  be  used,  or  Carbutt's  "  Multum 
in  Parvo  "  is  an  excellent  lamp  (Fig.  77).  This  lamp  has  three 
doors  ;  in  one  is  a  2  x  2.5  dm.  plate  of  ruby  glass  ;  in  the  second 
is  a  plate  of  opal  glass,  covered  with  a  metal  door,  which  is  used 
for  examining  the  finished  negative  ;  and  the  third  opening  con- 
tains a  clear  glass  through  which  the  white  light  comes  when  the 
lamp  is  used  for  contact  printing  in  making  lantern  slides. 

Development  pointers.  —  "i.  Development  containing  more 
alkali  produces  more  detail  in  the  shadows  and  lessens  intensity 
of  the  high  lights,  which  causes  more  softness  in  the  negative. 
Suitable  for  underexposed  plates. 

"2.  Extra  quantities  of  pyrogallic  acid,  eikonogen,  metol,  or 
hydrochinon  quickly  intensify  the  high  lights. 

"3.  Developer  diluted  with  water  slows  the  action  of  the 
chemicals  and  gives  the  shadows  more  chance  to  work  through 
before  the  high  lights  have  gained  their  strength,  and  is  recom- 
mended for  overexposed  plates. 

"  4.  There  is  no  such  thing  as  perfectly  safe  light,  and  the 
illumination  in  the  dark  room  must  be  carefully  looked  after. 
The  best  and  safest  light  for  ordinary  plates  and  the  eyes  is  a 
combination  of  ruby  and  orange  glasses. 

"5.  Dust  the  plate  slowly,  so  that  electric  currents  may  not 
be  generated  and  the  dust  become  electrified  and  render  the 
cleaning  of  the  surface  almost  impossible. 

"6.  Thorough  fixing,  thorough  washing  followed  by  quick  dry- 
ing in  the  shade,  will  insure  permanency  and  fine  printing  qualities. 

"7.  Do  not  expose  the  negative  to  white  light  until  all  trace 
of  the  bromide  of  silver  has  disappeared  from  the  back.  As  long 
as  there  is  any  white  cloudiness  on  the  back  keep  the  plate  in 
the  hyposulphite  of  soda  bath." 

Defects  in  the  Negative.  —  The  following  points  will  be  service- 
able to  the  beginner  in  determining  what  is  the  matter  with  an 
inferior  negative  and  what  steps  are  required  to  correct  the 
matter :  — 


I 


DEVELOPMENT   POINTERS  185 

"  i.  Foggy.  Caused  by  overexposure ;  white  light  entering 
the  camera  or  the  dark  room ;  too  much  light  during  develop- 
ment;  decomposed  chemicals  used  in  development;  introduc- 
tion of  hyposulphite  of  soda  into  the  developer ;  developer  too 
warm,  or  containing  too  much  carbonate  of  soda  or  potassium. 

"  2.    Weak  with  clear  shadows.     Underdevelopment. 

"3.    Too  strong  with  clear  shadows.     Underexposure. 

"  4.  Weak  with  plenty  of  detail  in  shadows.  Want  of  intensity 
caused  by  overexposure.  Shorter  exposure  with  longer  develop- 
ment will,  in  most  cases,  produce  intensity,  and  an  addition  of 
more  stock  solution  to  development  will  seldom  be  necessary. 

"5.  Fine  transparent  lines.  Too  stiff  brush  in  cleaning 
plates  before  placing  in  the  plate-holder. 

"  6.  Transparent  spots  and  pinholes.  Dust  on  the  plate  or 
in"  the  camera,  or  scum  on  old  developer,  or  air  bubbles  while 
developing.  Developer  must  be  dean. 

"7.  Crystallization  on  negative  and  fading  of  the  image. 
Hyposulphite  of  soda  not  well  washed  out  of  film  after  fixing. 

"  8.  Yellow  negatives.  Not  using  enough  sulphite  of  soda, 
or  the  article  is  old  and  decomposed. 

"9.  Mottled  negative.  Precipitation  from  fixing-bath  con- 
taining alum,  if  the  solution  is  old  and  turbid." 


CHAPTER   XIII 

MAKING   THE    POSITIVES 

A  POSITIVE  is  just  the  reverse  of  the  negative  and  represents 
the  view  in  its  complete  and  natural  condition.  Where  the 
shading  is  shown  in  the  negative  by  the  deposit  of  the  silver 
salts,  the  white  and  the  gradations  into  the  shadow  are  given 
in  the  positive.  Perfectly  clear  glass  on  the  negative  will  give 
deep  shadow  on  the  positive.  If  therefore  a  first-class  negative 
is  secured  it  is  not  a  difficult  matter  to  make  a  first-class  positive 
from  it,  but  if  the  work  of  exposing  the  sensitive  plate  and  its 
development  has  not  been  skilfully  done  the  negative  is  often 
too  poor  to  spend  any  time  in  making  positives  from  it,  and  it 
would  be  best  to  expose  another  plate  for  another  and  better 
negative,  if  the  object  is  near  and  of  easy  access.  Sometimes, 
however,  it  is  impossible  to  make  another  exposure  on  the  special 
view  and  we  are  compelled  to  attempt  to. make  our  positives 
from  an  inferior  negative  or  forego  having  pictures  of  the  scene. 
Under  these  conditions  some  little  ingenuity  and  skill  must  be 
exercised  to  obtain  a  picture  that  is  even  passable.  There  is  no 
excuse,  however,  for  using  an  inferior  negative  in  photo-micro- 
graphical  work,  because  the  student  can  readily  repeat  his  work 
until  he  does  obtain  a  good  negative,  since  the  apparatus  and  the 
object  are  located  near  the  dark  room  and  the  question  of  time 
simply  becomes  the  matter  for  consideration. 

Photographers  recognize  two  chief  kinds  of  positives :  — 

1.  Those  made  on  paper  and  cloth. 

2.  Those  made  on  glass. 

The  first  class  may  be  subdivided  into  :  — 
i.    Plain  and  albumenized  paper,  sensitized  with  silver  salts. 
These  papers  are  known  in  the  market  by  such  names  as  "  Disco," 

1 86 


MAKING  THE   POSITIVES,  l8/ 

"  Solio,"  "  Aristo-platino,"  "  W.  C.  Platinotype,"  etc.  These  are 
termed  by  photographers  "  printing-out  papers."  They  are 
made  by  exposing  the  sensitized  paper  under  the  negative  to  the 
sunlight  and  then  afterward  treating  them  with  chemicals  to 
fix  the  picture  and  tone  it  to  the  desired  color. 

2.  Slow  bromide,  or  developing  papers.     These  are  made  by 
exposing  the  sensitized  paper  under  the  negative  before  a  gas 
or  other  lamp  light  and  then  developing  the  picture  with  the 
same   chemicals  and  in  the  similar  manner  as  in  making  the 
negative,  until  the  image  comes  out  and  is  fixed  on  the  paper  in 
the  tone  desired.     The  trade  names  for  these  papers. are  "  Argo," 
"  Dekko,"  "  Nepera,"  "  Velox,"  "  Vinco,"  etc. 

3.  Ferro-Prussiate,  or  blue  papers. 

The  second  class  may  be  divided  into :  — 

1.  Lantern  slides. 

2.  Window  transparencies. 

To  make  positives  on  the  albumenized  paper,  proceed  as  fol- 
lows. This  paper  already  sensitized  may  be  purchased  from 
any  first-class  dealer  in  photographic  goods,  but  it  may  also  be 
made  in  the  laboratory  if  used  up  within  a  few  hours.  It  is 
difficult  to  make  this  character  of  paper  so  that  it  will  keep  for 
several  months,  but  the  brands  made  for  sale  are  manufactured 
under  a  secret  formula  which  preserves  the  sensitive  surface  for 
some  time. 

Select  the  proper  size  of  printing-frame  (see  Fig.  83)  to  suit 
the  size  of  negative,  place  the  negative  in  it  with  the  film  side 
up,  and  place  the  paper  in  contact  with  its  sensitive  surface  in 
touch  with  the  film  of  the  negative,  attach  firmly  the  back  of  the 
printing-frame  and  expose  the  glass  side  to  the  light  reflected 
from  the  sky,  if  the  negative  is  weak,  or  if  very  strong  to  the 
direct  light  from  the  sun.  Permit  the  action  of  the  light  to 
continue  until  the  picture  comes  out  slightly  deeper  than  the 
finished  picture  should  be,  because  the  treatment  with  the  chemi- 
cals will  somewhat  fade  the  image  and  allowance  must  be  made 
for  this.  Putting  the  sensitive  paper  under  the  negative  must 
be  done  in  a  portion  of  the  room  where  the  light  is  not  very 


1 88  BIOLOGICAL   LABORATORY   METHODS 

strong,  and  when  the  back  of  the  printing-frame  is  raised  to 
examine  the  progress  of  the  printing  turn  your  back  to  the 
light  and  expose  the  paper  off  of  the  negative  as  short  a  time 
as  possible. 

When  the  printing  has  been  carried  far  enough,  take  the  paper 
out  of  the  frame  and  immerse  in  a  vessel  of  water  for  a  minute 
so  that  a  portion  of  the  soluble  silver  salts  may  be  dissolved  out, 
and  then  transfer  to  the  following  toning-bath :  — 

Chloride  of  gold  I  grain  (0.06  gramme) 

Acetate  of  sodium      .         .         .         -3°  grains  (2.0  grammes)^ 
Water 8  ounces(24O.o  cc.) 

This  solution  must  not  be  used  at  once  but  must  be  allowed 
to  stand  for  about  24  hours.  Let  the  prints  remain  in  the  ton- 
ing-bath  until  they  assume  the  tone  desired  and  then  place  them 
in  the  fixing-bath,  which  is  prepared  as  follows :  — 

Hyposulphite  of  soda  ....       2  ounces  (60  grammes) 
Water 48  ounces  (1440  cc.) 

Fixing  in  this  bath  for  about  fifteen  minutes  will  suffice  to 
make  the  picture  relatively  permanent.  Do  not  permit  the 
prints  to  come  in  contact  with  each  other  while  fixing,  but  it 
will  be  best  to  keep  them  in  motion  for  the  finest  results.  After 
fixing  wash  them  in  running  water  for  half  an  hour,  or  in  six 
changes  of  water  after  soaking  in  each  change  for  five  minutes. 
It  is  absolutely  necessary  that  the  hyposulphite  of  soda  be 
entirely  eliminated  from  the  fibres  of  the  paper,  or  otherwise 
the  print  will  .soon  fade  and  turn  yellow.  Dry  after  washing 
thoroughly  by  spreading  the  papers  on  clean  blotting-paper  with 
the  picture  up,  and  then  store  them  away  until  ready  to  mount 
on  the  cardboard.  If  the  mounting  on  the  card  is  to  be  done 
at  once  proceed  as  follows  :  Trim  the  prints  to  the  size  and  shape 
desired  and  place  them  on  a  clean  glass  plate  with  the  picture 
side  down,  one  print  above  another,  until  all  have  been  trimmed 
and  thus  transferred  from  the  water  to  the  glass  plate.  Press 
out  the  water  with  the  hand  or  a  rubber  roller,  such  as  is  used 


MAKING  THE   POSITIVES  189 

by  photographers,  and  rub  over  the  top  print  a  thin  film  of  paste 
free  from  all  lumps  and  solid  matter.  Catch  the  paper  at  op- 
posite corners  and  bring  in  contact  with  the  card,  permitting  the 
centre  of  the  paper  to  touch  first  and  then  lowering  each  of  the 
corners  until  the  entire  portion  of  the  print  is  in  contact  with 
the  card.  Place  a  blotter  over  the  print  and  press  into  close 
union  with  the  card  by  moving  the  fingers  from  the  centre  to 
the  edges  of  the  paper.  Or  use  a  rubber  roller  which  is  made 
for  the  purpose.  When  the  mount  is  dry  the  paper  will  have 
contracted  and  all  wrinkles  will  disappear  and  the  surface  of  the 
paper  will  be  smooth. 

The  method  used  in  making  the  positives  with  the  developing 
papers  mentioned  under  the  second  subdivision  on  page  187  is 
somewhat  different  from  the  one  just  described.  These  develop- 
ing papers  are  much  more  rapid  in  their  work  than  the  printing- 
out  papers,  because  the  surfaces  are  more  sensitive  to  the  action 
of  light.  We  therefore  expose  them  before  a  lamp  instead  of 
the  direct  sun  rays.  The  same  kind  of  printing-frame  is  used 
with  these  as  in  the  case  of  the  papers  just  described.  Do  all  the 
work  in  the  dark  room  with  ruby  light  until  the  papers  are 
placed  under  the  negatives  and  until  ready  to  make  the  expos- 
ure. Stand  the  printing-frame  a  short  distance  from  the  lamp, 
the  distance  depending  on  the  character  of  the  negative  and  the 
strength  of  light,  and  then  permit  white  light  to  fall  on  the  glass 
side  of  the  negative  for  a  few  seconds,  from  ten  to  thirty,  de- 
pending also  on  the  density  of  the  negative  and  strength  of 
light.  Cover  the  white  light  and  proceed  to  develop  the  picture 
in  the  same  manner  as  that  used  in  making  the  negative,  viz. : 
Place  the  sensitive  paper  after  exposure  in  a  developing  pan 
with  the  sensitive  side  up  and  flood  over  it  a  developing  solution 
recommended  by  the  manufacturer  of  the  paper,  and  permit  this 
developer  to  act  until  the  picture  comes  out  clear  and  slightly 
deeper  in  shade  than  it  should  be  when  finished,  and  then 
fix  in  the  hyposulphite  bath  for  ten  or  fifteen  minutes.  Wash 
for  a  half-hour  in  running  water  to  extract  all  the  hyposul- 
phite of  soda  and  then  mount  in  the  manner  already  described 


BIOLOGICAL  LABORATORY   METHODS 

above.  To  illustrate  this  method  of  making  positives  with 
the  developing  papers  we  will  take  the  "  Vinco  "  through  the 
process. 

Expose  before  a  gas  lamp,  in  the  manner  described  above, 
and  develop  in  the  following  solution :  — 

Water 600  cc. 

Metol 2  grammes 

Hydrochinon      .......  ^  grammes 

Sulphite  of  soda  (granulated)     ....  7  grammes 

Carbonate  of  soda  (granulated)           ...  7  grammes 

Potassium  bromide T2o  grammes 

If  the  negative  is  vigorous  dilute  with  one-half  water ;  for  soft 
detail  negatives  shorten  the  exposure  and  use  the  full  strength 
of  the  developer.  After  the  picture  comes  out  to  the  degree  of 
shade  slightly  beyond  that  required  for  the  finished  picture, 
arrest  the  development  by  placing  the  print  in  the  following 
short  stop  and  hardener  :  — 

Table  salt  .         ,.    •    .         .         .         .         .         30  grammes 

Powdered  alum          .          .....         30  grammes 

Water        .         .         .         ....  ...        .       960  cc. 

One  minute  in  this  solution  will  suffice  ;  rinse  and  transfer  to 
the  fixing-bath  :  — 

Hyposulphite  of  soda 240  grammes 

Sulphite  of  soda  (granulated)    ....  30  grammes 

Sulphuric  acid 4  cc. 

Water 960  cc. 

Dissolve  the  sulphite  of  soda  and  hyposulphite  of  soda  in 
separate  vessels  and  add  the  sulphuric  acid  to  the  sulphite  of 
soda  solution  and  then  mix  the  two  solutions  together. 

Permit  the  prints  to  remain  in  the  fixing-bath  for  fifteen 
minutes,  and  then  bring  out  in  the  white  light  and  wash  in 
running  water  for  a  half-hour  until  all  the  hyposulphite  of  soda 
is  eliminated.  During  the  fixing  keep  the  prints  separate  so 
that  discoloration  will  not  take  place. 


MAKING   THE   POSITIVES  IQI 

Lantern  Slides :  How  to  make  them,  and  the  Use  of  the  Lantern 
for  projecting  Photo-micrographs  on  the  Screen 

The  sensitive  plates,  used  for  making  the  lantern  slides,  are 
coated  with  a  gelatin  bromide  emulsion  which,  when  exposed 
to  light  through  a  negative,  and  developed  according  to  direc- 
tions, will  give  an  attractive  positive  firmly  adhering  to  the 
glass.  Lantern  slides  of  a  high  grade  of  excellence  may  also 
be  made  by  the  use  of  the  "  wet  plates,"  which  of  course  must 
be  coated  with  the  sensitive  emulsion  just  before  using.  This 
is  the  old  method,  and  is  well  understood  by  the  professional 
photographer,  and  was  the  only  method  for  making  slides  before 
the  "  dry  plate  "  came  into  such  general  use.  It  requires  some 
experience,  however,  to  prepare  the  plate  properly,  and  there  is 
more  trouble  in  manipulating  it  in  the  hands  of  the  amateur. 
When  carefully  handled  the  dry  plates  give  brilliant  and  fine 
results,  and  they  are  to  be  recommended  to  the  student. 

The  dry  plates  are  sent  out  by  the  manufacturers  in  light-tight 
boxes,  one  dozen  in  each  box.  The  operator  should  obtain  a 
negative  box  the  proper  size,  and  fill  it  with  the  plates  after 
they  have  been  dusted  with  a  camel's-hair  brush.  This  transfer 
must,  of  course,  be  done  in  the  dark  room.  The  negative  box 
will  hold  twenty-four  plates. 

The  lantern  slides  are  made  by  either  one  of  the  following 
methods  :  — 

1.  The  contact  method. 

2.  Photographing  the  negative  by  means  of  a  camera. 

The  first  method  is  only  feasible  when  the  negative  has  been 
made  near  the  size  of  the  picture  to  be  transferred  on  the  posi- 
tive glass,  and  the  work  is  accomplished  as  follows  :  Mix  a 
sufficient  quantity  of  the  developer,  in  accordance  with  instruc- 
tions contained  on  page  179,  light  the  lamp,  and  exclude  all  rays 
from  the  dark  room  except  those  which  come  through  the  ruby 
glass  of  the  lamp.  Place  a  negative,  film  side  up,  in  a  deep 
printing-frame,  take  one  of  the  sensitive  plates  from  the  box 
and  lay  it  on  the  negative  with  film  in  contact  with  film,  place  a 


I Q2        BIOLOGICAL  LABORATORY  METHODS 

piece  of  thick  felt  on  the  back  of  the  sensitive  plate  to  exclude 
all  reflected  rays  of  light  from  the  rear  of  the  printing-frame,  and 
then  put  on  the  hinged  back  and  clamp  it  into  place.  Now  stand 
the  frame  on  edge  about  4.6  or  6.1  dm.  from  the  lamp  with  the 
negative  toward  the  light,  cover  the  frame  with  an  opaque 
cloth,  or  interpose  a  black  card,  and  then  open  the  door  of  the 
lamp,  so  that  the  white  light  may  be  projected  toward  the  print- 
ing-frame, raise  the  cloth  or  cover  for  a  few  seconds  (the  time 
depending  upon  the  distance  between  the  light  and  negative, 
strength  of  light,  and  density  of  negative)  until  the  sensitive 
plate  is  sufficiently  affected,  when  the  light  is  cut  off.  The 
plate  is  now  transferred  to  the  developing  pan  with  the  film  side 
up,  and  the  developing  solution  quickly  poured  over  it.  If  the 
picture  comes  up  rapidly,  the  exposure  has  been  too  long,  and 
another  trial  should  be  made.  There  is  but  little  advantage 
gained  in  trying  to  doctor  an  underexposed  or  very  much  over- 
exposed plate.  It  is  cheaper,  and  best  in  all  respects,  to  ex- 
pose another  plate,  either  prolonging  or  shortening  the  time  of 
exposure,  as  the  case  may  demand,  until  the  image  begins  to 
make  its  appearance,  not  sooner  than  twenty  seconds,  and  con- 
tinues to  come  out  gradually  and  slowly.  The  development 
must  not  be  stopped  until  the  high  lights  just  begin  to  tinge, 
and  all  the  details  of  the  picture  are  well  out,  and  distinct  con- 
trasts exist  between  the  high  lights  and  deep  shadows,  with 
gradual  blending  between  them.  The  plate  is  now  washed  and 
placed  in  a  fresh  fixing-bath  prepared  according  to  the  formula 
given  on  page  180.  It  must  remain  in  this  bath  until  all  the  white 
cloudiness  has  disappeared  entirely  from  the  back,  when  the  plate 
can  be  taken  out  into  the  white  light  and  examined  with  safety. 
The  positive  is  considered  to  be  first-class  if  it  will  show  by  trans- 
mitted light,  when  held  up  between  the  eye  and  the  window, 
perfectly  clear  glass  for  the  high  lights  and  passing  by  imper- 
ceptible gradations  to  the  deeper  shadows,  and  if  the  tone  is  of  a 
warm  purplish  black.  The  deep  shadows  must  not  be  so  opaque 
as  to  prevent  the  outlines  of  the  flame  of  a  lamp  from  being  seen 
when  the  slide  is  held  between  the  observer  and  the  light. 


MAKING  THE   POSITIVES  193 

Carefully  wash  the  slide  in  the  same  manner  described  for  the 
negative  and  swab  off  gently  with  a  wad  of  soft  cotton  to  take 
off  all  extraneous  particles  which  may  be  adhering  to  the  film, 
and  stand  in  a  rack  to  dry. 

To  make  the  slide  by  the  second  method,  or  photographing 
through  the  camera,  it  is  necessary  to  have  a  camera  with  an 
extra-long  bellows.  The  negative  is  suspended  in  front  of  a 
window  with  a  northern  exposure,  if  possible,  and  the  camera 
is  carefully  adjusted  at  a  suitable  distance  from  the  negative,  so 
that  the  image  projected  on  the  ground-glass  plate  will  be  the 
desired  size  for  the  slide.  The  front  of  the  camera  should  be 
parallel  with  the  surface  of  the  negative,  and  the  optical  axis  of 
the  lens  must  strike  the  centre  of  the  picture.  If  the  negative 
is  smaller  than  desirable,  reverse  the  lens  of  the  camera,  and  the 
enlargement  of  the  image  is  produced  on  the  ground  glass.  A 
little  ingenuity  in  manipulating  the  apparatus  will  soon  produce 
the  results  sought  for  by  the  operator.  As  soon  as  all  portions 
of  the  apparatus  have  been  properly  adjusted,  the  distance  be- 
tween the  negative  and  the  camera  is  covered  with  a  dark 
opaque  cloth  to  exclude  all  light  except  that  which  comes 
directly  through  the  negative,  and  the  operator  is  then  ready  to 
substitute  the  plate-holder  containing  the  sensitive  plate  for  the 
ground  glass  and  make  the  exposure.  Before  pulling  out  the 
slide  covering  the  plate,  see  that  the  lens  has  the  proper  dia- 
phragm and  is  covered  with  a  cap.  The  experience  of  the 
author  has  demonstrated  that  the  diaphragm  f  16  and  the  light 
coming  from  a  clear  northern  sky,  with  Carbutt's  "gelatino- 
albumen  "  plate,  fifteen  to  twenty  seconds,  will  yield  brilliant 
lantern  slides.  There  are  the  same  factors,  however,  to  be  con- 
sidered here  which  were  mentioned  in  the  case  of  the  time  of 
the  exposure  in  making  the  negative.  (See  page  177.)  The 
experience  of  the  operator  must  determine  these  questions  after 
he  has  become  familiar  with  the  qualities  of  his  negative  and 
the  character  of  the  light. 

The  after  treatment  of  the  plate  is,  in  all  respects,  the  same 
as  that  described  under  the  first,  or  contact,  method.  When 
o 


IQ4  BIOLOGICAL  LABORATORY   METHODS 

the  slide  is  thoroughly  dry  after  washing,  the  film  should  be 
covered  with  a  suitable  varnish  made  for  the  purpose  (usually 
colorless  gums  dissolved  in  benzol),  so  the  moisture  cannot 


FiG.  93.  —  Mat  for  Lantern  Slide. 

injure  the  slide  in  the  future.     Moreover,  the  varnish  benefits  the 
slide  in  cleaning  it  up  and  brightening  the  picture  throughout. 

To  mount  the  slide  for  permanent  preservation,  take  a  thin 
glass  plate,  perfectly  free  from  all  flaws  and  well  cleaned,  and 
cover  the  film.  Between  these  two  glass  plates  insert  the  mat 
illustrated  in  Fig.  93,  and  then  bind  the  plates  together  by  means 


MAKING  THE   POSITIVES  195 

of  a  narrow  strip  of  gummed  paper  three-eighths  of  an  inch  wide 
and  fifteen  inches  long.  Lay  this  strip  on  the  table  and  wet 
with  a  dampened  sponge  three  or  four  inches  at  one  end.  Take 
the  slide,  with  the  covering  glass  and  mat  in  position,  and  press 
one  of  the  edges  in  contact  with  the  wet  slip;  invert  the  slide, 
resting  the  opposite  edge  on  the  table,  and,  by  means  of  the 
fingers,  bring  the  damp  portions  of  the  slip  in  close  contact  with 
the  glass.  Proceed  to  dampen  the  gum  and  adhere  the  slip 


FIG.  94.  —  Box  for  Lantern  Slides. 

until  all  four  edges  of  the  slide  have  been  covered.  While  per- 
forming this  operation  keep  the  fingers  dry,  if  good  work  is  de- 
sired. The  label  is  placed  on,  and  then  the  slide  is  ready  for 
the  lantern. 

Figure  94  is  an  excellent  box  for  holding  the  slides  while 
they  are  being  put  through  the  lantern,  and  it  will  also  serve  a 
fine  purpose  in  preserving  them  from  the  action  of  dust  when 
stored  away. 

The  lantern  and  its  requisites.  —  The  lantern  consists  of 
the  following  parts,  which  are  absolutely  essential  to  its  well 
working :  — 


196  BIOLOGICAL   LABORATORY   METHODS 

1.  The  radiant  or  lamp. 

2.  The  condensers,  for  collecting  or  concentrating  the  light 
on  the  slide. 

3.  The  slide  carrier. 

4.  The  body  of  the  lantern,  which  consists  of  a  metal  box  or 
an  extension  bellows. 

5.  The  projecting  system  or  lens  for  projecting  the  rays  which 
pass  through  the  slide  on  to  the  screen  and  produce  the  enlarged 
picture. 

The  description  of  the  light  suitable  for  the  lantern  has  already 
been  given  in  Chapter  XI,  so  that  the  student  is  referred  to  that 
part  of  the  book  for  information  on  this  point.  What  has  been 
said  there  in  reference  to  illuminating  the  object  for  micro- 
photographing  is  equally  true  in  regard  to  the  character  of  the 
light  for  projection  through  the  lantern. 

There  are  several  first-class  lamps,  both  oxy-hydrogen  and 
electric,  on  the  market  which  have  been  especially  designed  for 
the  lantern,  and  the  principles  governing  them  being  so  nearly 
the  same,  the  description  of  one  will  give  a  clear  idea  of  the 
others.  On  page  168  is  illustrated  and  described  a  lamp  which 
the  author  has  been  using  for  several  years  with  considerable 
satisfaction,  for  the  micro-photographic  outfit  and  for  the  lantern. 
This  lamp,  or  one  like  it,  will  be  found  indispensable  in  the 
laboratory  when  it  is  desired  to  project  on  the  screen  a  slide  of 
the  object  which  is  being  studied,  either  in  the  shape  of  a  photo- 
graph on  glass  or  a  projection  of  an  image  direct  from  the  object 
itself,  by  means  of  the  microscope  attachment  to  the  lantern. 
Figure  95  is  an  illustration  of  how  this  application  of  the  electric 
lamp  and  the  lantern  with  the  microscope  attachment  may  be 
used.  The  object  is  placed  on  the  stage  at  a  and  the  lamp  b 
transmits  the  light  through  the  lantern  c,  to  the  object,  and  the 
objective  o  magnifies  the  image  and  sends  it  to  the  lens  //, 
where  it  is  projected  to  the  screen  greatly  enlarged. 

The  oxy-hydrogen  lamp  is  made  to  attach  to  this  outfit  in  the 
same  manner,  if  it  is  desired  to  use  the  lime  light  instead  of  the 
electric  light. 


198 


BIOLOGICAL   LABORATORY    METHODS 


Where  there  are  two  lanterns  in  the  laboratory,  beautiful 
effects  are  secured  by  producing  what  is  termed  dissolving 
views.  This  is  accomplished  by  placing  the  lanterns  near  each 
other,  so  that  the  beam  of  light  thrown  from  each  will  cover  the 
same  portion  of  the  screen ;  and,  if  the  oxy-hydrogen  jets  are 
used,  the  key  illustrated  in  Fig.  96  is  so  connected  with  the 
oxygen  and  hydrogen  cylinders  that  the  gases  will  pass  through 


FlG.  96.  —  Dissolving  Key. 

the  key  and  may  be  transmitted  first  to  one  of 
the  lanterns  and  then  to  the  other  at  the  will 
of  the  operator.  The  effects  on  the  screen 
are  very  beautiful  when  one  picture  dissolves 
off  as  the  other  grows  into  full  strength. 

The  care  of  the  lantern.  —  Like  all  other  delicate  apparatus 
the  lantern  is  liable  to  serious  damage  unless  carefully  protected 
from  dust  and  sudden  changes  of  temperature.  When  the  lamp 
is  lighted  the  flame  should  be  kept  rather  low  until  the  con- 
densers are  gradually  heated.  For  this  purpose  it  is  best  to 
turn  on  the  light  ten  or  fifteen  minutes  before  the  pictures  are 
projected  on  the  screen,  and  then  the  light  can  be  raised  to  its 
fullest  intensity  without  so  much  danger  of  breaking  the  con- 
densers and  other  glass  lenses.  An  excellent  protection  against 
flying  particles  of  highly  heated  lime  and  carbon  is  a  piece  of 


MAKING  THE   POSITIVES  199 

clear  mica  interposed  between  the  light  and  the  lenses.  The 
same  care  should  be  manifested  in  turning  off  the  light  to  pre- 
vent the  glasses  from  cooling  too  quick,  because  the  unequal 
and  rapid  contraction  will  often  cause  them  to  break.  When 
not  in  use  the  lantern  should  be  carefully  covered  with  a  thick 
cloth  to  protect  all  parts  of  it  from  dust.  Whenever  it  is  neces- 
sary to  clean  any  portion  of  the  glass  surface,  the  best  material 
to  use  is  Japanese  paper,  because  it  contains  no  particles  of  grit 
and  is  very  soft  and  has  considerable  absorbent  power  for  the 
moisture  frequently  condensed  on  the  surfaces  of  the  lenses. 
The  rubbing,  however,  should  be  gentle,  that  no  scratching  may 
be  done.  Rub  the  glass  as  little  as  possible  ;  it  is  much  better  to 
protect  them  from  all  particles  of  dust  and  sudden  changes  of 
temperature. 

The  light  must  be  centred  on  the  screen  before  the  slide  is 
put  in  the  lanterns  ;  so  that  a  perfectly  white  light,  free  from  all 
shadows  and  colors,  will  be  formed.  This  is  done  by  adjusting 
the  lamp  in  three  ways  :  vertically,  laterally,  and  from  or  toward 
the  condensers.  This  operation  is  very  quickly  accomplished 
when  once  learned.  It  is  only  necessary  thereafter  to  adjust 
the  front,  projecting  lenses  as  each  slide  may  require. 

The  fact  must  be  borne  in  mind,  in  determining  the  distance 
between  the  screen  and  the  lantern,  that  the  nearer  the  lantern 
is  placed  the  smaller  will  be  the  picture  ;  and,  as  a  general  rule, 
also,  the  greater  the  size  of  the  picture  the  less  will  be  the 
brightness  of  the  disk  of  light  on  the  screen. 


CHAPTER   XIV 

APPARATUS    AND    METHODS    IN    THE    STUDY   OF 
BACTERIOLOGY 

IN  the  study  of  bacteria  it  becomes  necessary  to  adopt  meas- 
ures to  determine  the  peculiar  habits  and  methods  of  growth  of 
these  minute  forms  in  order  that  the  experimenter  may  arrive  at 
conclusions  concerning  their  life  history.  These  microscopic 
forms  must,  therefore,  be  cultivated  in  the  laboratory,  and  the 
steps  of  growth  carefully  noted  and  studied.  In  order  to  carry 
on  this  work  to  a  successful  conclusion  the  laboratory  must  be 
supplied  with  certain  kinds  of  apparatus  and  chemicals  not 
mentioned  in  the  other  portions  of  this  work. 

The  special  disease,  or  bacteria,  must  be  developed  by  breed- 
ing experiments  in  flasks  or  test-tubes,  and  all  the  utensils  and 
apparatus  used  in  their  cultivation  must  be  carefully  sterilized, 
so  that  no  other  organisms  will  enter  the  breeding  vessels  and 
confuse  the  observations  on  the  special  germ  under  examination. 
The  intention  of  this  chapter  is  to  give  in  detail  the  steps  to  be 
taken,  and  the  apparatus  and  chemicals  required  to  conduct  the 
culture  experiments  in  a  well-equipped  laboratory. 

The  vessels  may  be  sterilized  by  heating  to  a  high  degree 
(160°  to  200°  C.),  until  all  living  organisms  which  may  be  in  the 
air  of  the  vessel,  or  on  its  inner  surfaces,  are  destroyed,  and  the 
vessel  must  then  be  kept  closely  stopped  with  cotton-wool  which 
has  also  been  sterilized.  The  nutrient  fluids  which  are  to  be 
used  in  the  culture  experiments,  or,  in  other  words,  which  are  to 
be  the  foods  for  the  growing  germs  during  their  development, 
must  also  be  treated  with  heat,  so  that  all  life  in  them  will  also 
be  entirely  destroyed  before  the  bacilli  to  be  studied  are  planted 
in  them.  This  is  accomplished  in  the  following  manner  :  — 

200 


BACTERIOLOGICAL  APPARATUS  2OI 

The  nutrient  substance  is  boiled  in  a  flask  a  short  time  each 
day  for  a  period  of  several  days,  and  the  vessel  must  be  closed 
with  the  cotton  stopper.  Fill  the  flask  about  one-half  full,  so 
that  in  the  boiling  the  cotton  will  not  get  wet.  Pull  out  the  cot- 
ton about  halfway  during  the  boiling,  and  after  finishing  heat- 
ing, push  in  the  cotton  again.  A  beaker  with  sterilized  cotton 
is  placed  over  the  mouth  of  the  flask,  and  the  vessels  are  put 
aside  over  night.  After  boiling  the  first  day  certain  bacteria 
are  destroyed,  but  there  are  other  spores  which  are  able  to 
stand  higher  temperatures  ;  and  after  permitting  the  flask  to 
stand  a  few  hours  after  each  boiling,  these  spores  will  have 
an  opportunity  to  germinate,  and  a  second  boiling  will  also  kill 
them.  Hence  the  reason  for  successive  heating  on  several 
different  days.  It  has  been  found  that  after  four  or  five  days  of 
treatment  in  this  manner  all  life  will  be  destroyed  in  the  nutrient 
fluid,  and  it  will  then  be  in  a  condition  for  the  transplanting  of 
the  special  disease  to  be  studied.  By  properly  inserting  the 
cotton-wool,  there  will  be  no  danger  of  infection  from  the  atmos- 
phere, as  long  as  the  cotton  remains  in  the  neck  of  the  flask. 
The  greatest  danger  comes  from  the  inner  surfaces  of  the 
flask,  which  may  have  microscopic  spores  lodged  thereon  be- 
fore the  cotton  was  inserted,  provided  the  flask  was  not 
properly  sterilized.  After  the  final  boiling  of  the  nutrient 
fluid,  and  after  standing  for  some  hours,  its  condition  will 
indicate  whether  or  no  the  substance  is  free  from  all  organ- 
isms, and  therefore  suitable  for  the  inoculation,  or  sowing,  with 
the  spores  under  treatment.  When  the  final  boiling  is  com- 
pleted, the  flask,  plugged  with  sterilized  cotton  and  covered 
with  the  beaker,  is  placed  in  the  incubator  and  left  there  in  a 
temperature  of  32°  to  38°  C.  for  one  to  three  weeks,  and  at  the 
end  of  that  period,  if  the  nutrient  fluid  remains  limpid,  it  may 
be  used  with  safety  and  with  the  assurance  that  all  bacilli  are 
destroyed. 

To  sterilize  vessels,  such  as  flasks,  beakers,  and  test-tubes, 
expose  them  for  a  while  to  a  strong  heat  from  a  Bunsen  burner. 
The  heat  must  be  applied  to  all  surfaces  thoroughly,  and  the 


202  BIOLOGICAL  LABORATORY   METHODS 

inner  surfaces  must  be  made  specially  hot.  While  in  a  heated 
condition  the  mouths  of  the  vessels  must  be  closed  with  the 
plugs  of  cotton  one  to  two  inches  long,  which  have  been  also 
sterilized.  Place  these  plugs  in  firmly  but  not  too  tight.  When 
a  number  of  vessels  are  to  be  sterilized  this  may  be  accomplished 
in  the  air-chamber,  where  a  heat  of  140°  to  150°  C.  is  sustained 
for  a  period  of  four  to  six  hours.  While  in  the  hot  condition 
the  vessels  are  taken  out,  one  at  a  time,  and  plugged  with  the 
sterilized  cotton.  They  are  then  replaced  in  the  air-chamber 
and  the  heat  repeated  for  several  hours. 

The  cotton  is  rendered  sterile  by  pulling  open  the  fibres  and 
exposing  them  to  a  temperature  of  150°  C.  in  the  air-chamber 
until  a  slight  charring  is  evident.  The  heat  is  repeated  for 
several  successive  days,  so  that  all  spores  which  may  germinate 
between  each  heating  will  be  destroyed.  Care  must  be  exer- 
cised to  heat  the  cotton  enough  but  not  so  far  as  to  cause  it  to 
become  brittle.  It  must  retain  its  pliable  condition,  although 
some  of  the  fibres  have  become  slightly  charred. 

By  observing  the  above  precautions  there  need  be  no  appre- 
hension on  the  part  of  the  experimenter  that  his  experiments 
will  be  vitiated  by  badly  sterilized  vessels  and  cotton. 

All  metal  instruments,  such  as  knives,  needles,  etc.,  should 
be  constructed  to  stand  a  strong  heat  without  injury.  And  be- 
fore use  they  should  be  subjected  to  the  flame  of  a  Bunsen 
burner. 

The  Apparatus  used  in  Bacteriology 

Hot-air  and  steam  sterilizers.  —  There  are  several  patterns  in 
the  market,  and  among  the  standard  makes  may  be  mentioned : 

1.  Pasteur's  hot-air  sterilizer. 

2.  Lautenschlaeger's  hot-air  sterilizer. 

3.  Bausch  &  Lomb's  hot-air  sterilizer. 

4.  Koch's  steam  sterilizer. 

5.  Arnold's  steam  sterilizer. 

6.  Autoclave,  or  steam  digester,  or  pressure  sterilizer.      Several 

of  the  leading  autoclaves  are  :  — 


BACTERIOLOGICAL  APPARATUS  2O3 

a.  Dr.  Pfungst's  autoclave. 

b.  Lautenschlaeger's  autoclave. 

c.  Bausch  &  Lomb's  autoclave. 

d.  Petri's  autoclave. 

These  sterilizers  may  be  classified  as  follows  :  — 

1.  Hot-air  sterilizers. 

2.  Steam  sterilizers. 

3.  Autoclaves  or  pressure  sterilizers. 

Hot-air  sterilizers.  —  This  apparatus,  Figs.  97  and  98,  consists 
of  a  metal  box  containing  shelves  and  covered  with  suitable  ma- 
terial to  prevent  heat  radiation.  The  heat  is  applied  by  means 
of  a  Bunsen  burner  at  the  base  of  the  apparatus.  Attached  to 


FIG.  97.  —  Hot-air  Sterilizer.     (B.  &  L,.j 


204        BIOLOGICAL  LABORATORY  METHODS 

this  sterilizer  are  also  thermometers  and  thermo-regulators,  so 
that  the  degree  of  heat  may  be  under  control  and  a  constant 
temperature  may  be  retained  as  long  as  desired.  The  hot  air 
circulates  not  only  over  the  bottom  of  the  apparatus  but  also  up 


FIG.  98.  — Hot-air  Sterilizer,  Lautenschlaeger's  Pattern. 

the  sides  and  over  the  top,  so  that  the  degree  of  temperature  is 
uniform  over  the  entire  instrument.  The  sterilizer  has  double 
sides  in  order  that  the  air  may  be  thus  circulated.  The  arrows 
in  the  illustration  show  which  way  the  gas  goes  when  the  heat  is 
applied  at  the  bottom  of  the  apparatus. 


BACTERIOLOGICAL  APPARATUS 


2O5 


Steam  sterilizers.  —  These  instruments  are  generally  used  to 
sterilize  nutrient  media,  and  for  filtering  fluids,  which  are  diffi- 
cult to  filter  except  when  hot.  The  apparatus  is  made  of  copper 
with  a  sterilizing  chamber  so  arranged  that  the  articles  placed 
in  it  are  subjected  to  a  current  of  steam.  This  instrument  is 
also  provided  with  thermometers,  and  the  whole  is  covered  with 


FIG.  99.  — Arnold's  Steam  Sterilizer. 

a  non-conducting  substance  to  prevent  heat  radiation.  The 
Arnold  form  of  the  steam  sterilizer  (Figs.  99,  100,  and  101)  is 
operated  by  placing  water  in  the  pan  or  lower  reservoir,  where 
it  passes  by  degrees  into  the  lowest  compartment  through  small 
openings  to  be  heated  by  the  burner  and  driven  into  steam.  The 
stream  rises  through  a  central  shaft  to  the  sterilizing  chamber, 
where  the  vessels  are  located.  The  action  is  almost  instantaneous 
because  the  water  film  in  the  lowest  compartment  is  so  thin  that 


206 


BIOLOGICAL  LABORATORY  METHODS 


FlG.  100.  —  Arnold's  Steam  Sterilizer.    Section. 


the  heat  causes 
it  to  boil  at  once 
and  the  steam 
rises  rapidly  into 
the  chamber 
around  the  ves- 
sels to  be  steril- 
ized. There  is 
thus  around  the 
vessels  a  constant 
temperature  of 
100°  C.  The  ex- 
cess of  steam  es- 
capes through  the 
cover  or  door. 

Autoclave,       or 
pressure  sterilizer. 

—  Some  spores  resist  the  action  of  heat  more  strenuously  than 

others,  and  it  becomes  necessary  to  apply  a  higher  degree  of 

heat  than  can  be  secured  with 

the   usual  form   of  hot-air  or 

steam  sterilizers,  at  the  ordi- 
nary atmospheric  pressure ;  so 

the  autoclave   (Fig.   102)  has 

been   devised   to   give   stearn 

under  high  pressure  with  tem- 
perature   raised    to    135°    C. 

Thirty  pounds'  pressure  to  the 

square   inch  can  be  obtained 

by  means  of   this  apparatus, 

which  has  been  found  to  be 

equivalent    to    134.6°    C.      A 

pressure-gauge  is  attached  to 

the  autoclave  to  measure  this  _ 

pressure,  and  an  escape-valve      FlG  IOI._Arnold-s  Steam  sterilizer. 

is  also  connected  so  that  the  Section. 


BACTERIOLOGICAL  APPARATUS 


207 


action  of  the  steam  may  be  under  control.      A  thermometer 
gives  the  temperature,  and  an  air-valve  is  placed  on  the  ap- 


FlG.  102. — Autoclave. 


paratus,  which  must  be  kept  opened  until  the  air  is  all  driven 
out  by  the  steam,  which  is  indicated  by  the  blowing  out  of  the 


208 


BIOLOGICAL  LABORATORY  METHODS 


steam.  This  autoclave  can  be  also  used  as  an  ordinary  steam 
sterilizer  at  the  normal  temperature  if  desired ;  so  that  if  the 
laboratory  possesses  an  autoclave  the  steam  sterilizers  may 


FIG.  103.  —  Laboratory  Incubator.     (B.  &  L.) 

be  omitted  from  the  list  of  apparatus  when  the  equipment  is 
purchased.  The  heat  is  applied  by  means  of  a  ring  Bunsen 
burner. 

The  autoclave  costs  more  than  either  the  hot-air  or  steam 


BACTERIOLOGICAL  APPARATUS 


2O9 


sterilizer,  but  the  laboratory  in  which  work  is  carried  on  in 
experimenting  with  plant  and  animal  diseases  cannot  well  afford 
to  do  without  it. 

Incubators.  —  These  instruments  (Fig.  103)  are  absolutely  essen- 
tial in  the  laboratory  where  culture  experiments  are  conducted. 


FIG.  104.  — Wire  Baskets. 

The  incubator  is  used  to  grow  the  bacteria  in  a  temperature  which 
is  under  control  by  a  thermo-regulator.  The  illustration  shows 
the  usual  form  to  be  found  in  the  large  laboratories.  There 
are  two  doors,  one  of  plate  glass  and  one  solid ;  both  are  close 
fitting,  so  that  they  are  practically  air- 
tight. The  heat  is  communicated  by 
means  of  a  Bunsen  burner  in  the 
lower  compartment,  and  this  burner  is 
of  the  double  Koch  safety  pattern.  The 
incubator  is  constructed  to  hold  water 
between  the  double  walls,  and  there  is 
also  a  hot-air  chamber.  The  heat  being 
under  perfect  control,  the  growing  cul- 
tures can  be  developed  under  known  and 
fixed  temperatures. 

The   shelves   are   used   to   hold   wire 


FIG.  105.  —  Wire  Basket 
containing  Culture  Tubes. 


baskets    in   which   the   test-tubes    containing   the   cultures    are 
stacked.     Figure   104  shows  two  forms  of  this  basket, 
p 


210  BIOLOGICAL   LABORATORY   METHODS 

Gas  pressure  regulators.  —  In  the  use  of  incubators  and  steril- 
izers it  is  of  the  utmost  importance  that  the  heat  should  be  regu- 
lated, and  this  must  be  accomplished  by  an  automatic  control  of 
the  flow  of  gas  to  the  burners.  The  gas  must  first  pass  through 
a  pressure  regulator  and  then  through  a  thermo-regulator  before 
it  reaches  the  burner.  There  are  several  instruments  on  the  mar- 
ket for  securing  these  ends.  The  description  of  Reichert's  given 
below  will  explain  the  general  principles  controlling  them  all. 


FIG.  106.  —  Reichert's  Thermo-regulator. 

Reichert's  thermo-regulator  (Fig.  106). — There  are  two  portions 
of  this  instrument,  a  hollow  T-shaped  piece,  in  which  one  end  of 
the  horizontal  section  is  open  and  the  other  end  is  closed,  a  tube 
containing  mercury  and  two  horizontal  orifices.  At  the  lower 
end  of  the  T-tube  are  two  small  holes,  one  at  the  point  and  the 
other  on  one  side.  The  instrument  is  placed  in  the  roof  of  the 
incubator  so  that  the  bulb  is  in  contact  with  the  water  or  extends 
in  the  interior  of  the  chamber,  and  the  gas  is  passed  through  a 
rubber  to  the  open  end  and  out  into  another  rubber  tube  leading 
to  the  burner.  When  the  desired  temperature  is  reached  the 
mercury  is  forced  up  the  tube  by  means  of  the  screw  until  the 
orifice  in  the  T-tube  is  closed,  excepting  the  small  orifice  on 
the  side,  when  the  flame  will  lower  and  the  heating  will  be 


FIG.  107.  —  Novey's  Thermo-regulator. 


NUTRIENT   MEDIA  211 

diminished.  The  mercury  will  then  recede  and  the  flow  of  gas 
will  again  increase,  and  in  this  way  the  temperature  will  be 
controlled  automatically. 

Dr.  F.  G.  Novey  has  recommended  a  change  in  Reichert's 
regulator  which  is  designed  to  prevent  the  passage  of  more  gas 
through  the  minimum  opening  than  is  required  to  maintain  the 
desired  temperature  (Fig.  107). 

Nutrient  Media  and  their  Preparation 

Nutrient  media  are  the  chemicals  used  in  the  laboratory 
devoted  to  bacteriological  research,  for  the  food  of  the  bacteria 
during  the  process  of  culture.  In  the  study  of  diseases  of  plants 
or  animals  it  becomes  necessary  to  grow  the  bacteria  under 
known  conditions  in  order  that  the  life-history  of  the  germ  may 
be  well  understood ;  and  to  do  this  work  satisfactorily  the  germ 
must  be  developed  in  a  medium  which  is  a  food  for  the  spores 
when  they  begin  to  germinate,  and  which  at  the  same  time  is 
sufficiently  transparent  so  that  the  experimenter  may  observe 
the  different  stages  of  growth. 

There  are  generally  three  kinds  of  these  nutrient  media,  viz. : 

1.  Nutrient  bouillon. 

2.  Nutrient  agar-agar. 

3.  Nutrient  gelatin. 

Several  other  substances  are  used  in  the  laboratory  as  food 
for  the  bacilli,  such  as  slices  of  potatoes,  carrots,  turnips,  blood 
serum,  etc.,  but  the  media  generally  used  are  the  three  men- 
tioned above.  In  the  preparation  of  these  nutrient  substances 
the  following  steps  must  be  taken. 

Nutrient  Bouillon.  —  Add  1000  cc.  of  water  to  500  grammes 
of  finely  chopped,  or  ground,  lean  beef,  freed  from  fat.  Place 
the  vessel  containing  this  beef  in  the  water-bath  and  warm 
gently  to  a  temperature  of  55°  to  60°  C.  for  the  period  of  an  hour, 
with  frequent  stirring.  If  the  temperature  goes  above  60°  C.  the 
albuminous  substances  will  coagulate,  which  will  somewhat 
interfere  with  the  final  clearing  of  the  fluid.  After  heating  for 
an  hour,  and  when  the  soluble  matter  of  the  beef  has  been  dis- 


212  BIOLOGICAL   LABORATORY   METHODS 

solved,  the  mass  is  strained  through  a  linen  cloth.  The  filtrate 
is  made  up  to  1000  cc.  by  the  addition  of  water.  Add  to  this 
fluid,  which  is  a  deep  red  color,  five  grammes  of  salt  and  ten 
grammes  of  a  i  per  cent  Witte's  peptone,  and  warm  until 
solution  is  complete,  but  taking  care  not  to  let  the  temperature 
go  below  60°  C.  Render  the  solution  slightly  alkaline  by  the 
addition  of  a  saturated  solution  of  sodium  carbonate ;  test  care- 
fully with  litmus-paper1  until  the  red  slightly  turns  blue.  Boil 
for  about  a  half-hour  and  filter.  Cool  and  distribute  in  Erlen- 
meyer  flasks  or  test-tubes  and  plug  with  cotton.  The  usual 
quantity  for  each  flask  is  250  cc.  Sterilize  for  fifteen  minutes 
in  the  steam  sterilizer  each  day  for  three  days,  before  inoculating 
with  the  germ  submitted  for  investigation. 

Nutrient  Agar-agar.  —  Agar-agar  is  a  seaweed  found  on  the 
coast  of  Japan,  and  is  sometimes  sold  under  the  name  of  Bengal 
isinglass  because  this  seaweed  is  also  found  near  Singapore. 
When  dissolved  in  water  it  yields  a  thick,  odorless  jelly. 

In  the  preparation  of  this  nutrient,  bouillon  is  used  as  the 
base.  The  agar-agar  is  added  for  the  purpose  of  solidifying  the 
mass  in  the  form  of  a  jelly.  Add  to  the  liter  of  bouillon  20 
grammes  of  agar-agar  which  has  been  finely  cut  up.  Place  the 
vessel  in  the  autoclave,  or  steam  sterilizer,  and  heat  until  the 
agar-agar  is  thoroughly  dissolved.  This  will  generally  take 
from  one-half  to  one  hour.  The  vessel  is  then  placed  in  the 
water-bath  at  a  temperature  of  60°  C.  until  the  solid  particles 
settle  to  the  bottom,  when  the  fluid  is  filtered  through  absorbent 
cotton.  The  filtering  of  this  nutrient  agar-agar  is  a  very  slow 
and  tedious  work.  Dr.  M.  P.  Ravenel  gives  the  following 
method  for  preparing  and  filtering  this  nutrient,  which  he  claims 
to  be  superior  to  any  other  method  both  in  the  time  saved  in 
filtering  and  in  the  character  of  the  fluid  obtained  : 2  — 

1  G.  W.  Fuller  titrates  with  phenolphtalein  in  place  of  litmus,  as  an  indicator  in 
adjusting  the  reaction  of  the  media.     Comparative  experiments  on  alkaline  media 
showed  that  water  bacteria  throve  best  on  those  which  contain  15  to  20  cc.  normal 
alkali  per  liter.     (Jour.  Amer.  Pub.  Health  Ass.  X,  1895.) 

2  Jour.  App.  Mic.,  Vol.  I,  p.  106. 


NUTRIENT   MEDIA  213 

"  To  make  one  liter  of  agar-agar,  take 

A.  Dried  peptone  (i%) 10  grammes 

Common  salt  (0.5%) 5  grammes 

Liebig  extract  (0.5%) 5  grammes 

Water 500  cc. 

"  Boil  for  three  minutes  and  neutralize. 

B.  Agar-agar  (1.2%) 12  grammes 

Water 500  cc. 

"  Chop  the  agar-agar  and  put  into  autoclave.  Run  the  auto- 
clave up  to  two  atmospheres  of  pressure,  giving  i2i.4°C.  of 
heat.  As  soon  as  this  pressure  is  reached,  turn  out  the  flame 
and  jallow  the  autoclave  to  cool  until  below  ioo°C.  before  open- 
ing. The  two  solutions  A  and  B  are  then  mixed,  cooled  to  60°  C., 
the  whites  of  two  eggs  beaten  in  50  cc.  of  water  added  and  well 
stirred  in,  and  the  whole  then  boiled  and  filtered  through  paper. 

"  The  whole  process  requires  only  an  hour  and  a  quarter  to 
an  hour  and  a  half,  and  the  result  is  a  most  excellent  jelly. 
Instead  of  white  of  egg,  blood  serum  may  be  used,  which  seems 
to  add  also  to  the  nutritive  value  of  the  medium.  Agar  made 
with  meat  extract  will  often  form  a  precipitate  during  the  ster- 
ilization, which  is  objectionable  if  one  wishes  to  use  it  in  the 
making  of  Esmarch's  roll-tubes. 

"  To  make  an  absolutely  and  permanently  clear  agar,  fresh 
meat  should  be  used  as  follows  :  — 

"  To  make  one  liter,  take 

A.  Chopped  meat 500  grammes 

Water .         .         500  cc. 

"  Mix  and  place  in  cool  place  over  night,  then  strain  through 
towel. 

B.  Agar-agar  (1.2%) 12  grammes 

Water 500  cc. 

"  Put  in  autoclave,  run  up  to  two  atmospheres  of  pressure, 
put  out  flame,  and  allow  to  cool  until  below  100°  C.  before  open- 
ing. Let  the  solution  of  agar  cool  still  further  to  about  75°  C.? 


214  BIOLOGICAL   LABORATORY   METHODS 

and  then  mix  A  and  B,  add  (i%)  10  grammes  dried  peptone  and 
(°'5%)  5  grammes  of  common  salt,  bring  to  a  boil  for  about 
three  minutes,  neutralize  and  filter.  The  product  is  an  abso- 
lutely clear  jelly  which  never  forms  a  precipitate.  The  whole 
process,  with  the  exception  of  the  time  the  meat  is  steeping, 
requires  only  about  one  hour  and  a  half.  In  both  of  the  above 
methods  the  filtration  is  very  quick  —  from  ten  to  twelve  min- 
utes for  the  liter.  I  never  use  a  hot-water  funnel,  but  wet  the 
filter-paper  with  boiling  water  immediately  before  pouring  in  the 
agar.  In  the  process  with  fresh  meat  the  clarification  is  effected 
by  the  coagulation  of  the  albumen  in  the  meat  water,  hence 
solution  B  must  not  be  added  to  A  until  cool  enough  to  avoid 
coagulation.  In  general  the  fresh  meat  is  to  be  recommended, 
and  the  process  is  easier  than  with  the  meat  extract,  though  the 
latter  has  the  advantage  of  cheapness  and  convenience,  since 
the  meat  extract  can  always  be  kept  on  hand,  and  the  time  lost 
in  soaking  the  fresh  meat  is  saved." 

Nutrient  gelatin.  —  The  bouillon  is  also  the  base  of  this 
nutrient.  In  its  preparation  1000  cc.  of  bouillon  are  taken, 
and  to  the  solution  are  added  5  grammes  of  common  salt,  100 
grammes  of  gelatin,  and  10  grammes  of  Witte's  peptone  (i%). 
Heat  is  applied  in  the  water-bath  until  the  gelatin  is  dissolved. 
The  fluid  is  then  rendered  slightly  alkaline  by  the  careful  addi- 
tion of  a  saturated  solution  of  carbonate  of  soda.  The  vessel 
is  again  placed  on  the  water-bath  and  the  water  in  the  bath 
boiled  for  one  hour.  The  albuminoids  in  the  nutrient  separate 
in  flakes,  when  the  fluid  is  filtered.  The  filtrate  must  have  the 
following  properties :  — 

1 .  Solidify  on  cooling. 

2.  Be  perfectly  clear  and  remain  so  after  heating   in   the 
sterilizer. 

3.  Show  slight  alkaline  action. 

The  nutrient  gelatin  and  nutrient  agar-agar  have  different 
melting-points,  and  this  fact  must  be  borne  in  mind  when  using 
them  for  culture  experiments.  The  first  melts  at  the  tempera- 
ture of  the  body,  while  the  latter  requires  a  temperature  as  high 


CULTURE   METHODS— INOCULATION  21$ 

as  boiling-point.  Some  bacteria  require  higher  temperatures 
for  their  growth  than  do  others,  and  in  such  cases  the  nutrient 
agar-agar  must  be  used. 

Potato  culture  method.  —  Select  a  sound  potato,  and  cut  off 
the  ends  and  core  with  an  apple-corer,  making  the  sections 
about  25  mm.  in  diameter.  Make  an  oblique  section  across 
the  centre  of  the  core,  thus  dividing  it  into  two  pieces.  Place 
in  running  water  for  a  time  to  prevent  discoloration,  and  then 
submit  to  the  action  of  a  dilute  alkali  solution  in  order  to  render 
the  pieces  uniform*  in  neutral  or  slightly  alkaline  reaction.  Now 
put  these  sections  of  the  potato  in  test-tubes  with  the  oblique 
faces  upward,  sterilize  an  hour  each  day  for  several  days  in  a 
steam  sterilizer,  and  then  inoculate  with  a  sterilized  knife  con- 
taining the  material  for  study. 

Blood  serum.  —  Wash  the  vessels  which  are  intended  to  re- 
ceive the  blood,  first  with  sublimate,  then  with  alcohol,  and 
finally  with  ether.  The  latter  is  allowed  to  evaporate.  The 
place  on  the  skin  of  the  animal  (sheep,  calf,  or  horse)  is  washed 
with  sublimate,  and  an  incision  is  made  with  a  sharp  sterilized 
scalpel.  The  first  outflow  of  the  blood  is  rejected,  and  then  the 
quantity  desired  is  received  in  the  vessels  as  above  prepared, 
and  the  glass  stoppers,  greased  with  vaseline,  are  replaced,  and 
the  vessels  are  quickly  transferred  to  ice,  where  they  remain  for 
twenty-four  to  thirty-six  hours.  After  the  clot  separates,  the 
clear  serum  is  drawn  off  into  sterilized  test-tubes,  which  are 
closed  with  cotton  plugs.  Now  sterilize  with  a  temperature  of 
80°  to  90°  C.  for  one  hour,  and  repeat  the  operation  for  six  suc- 
cessive days.  To  solidify  the  serum  raise  the  temperature  to 
100°  C.,  and  the  tubes  are  then  ready  for  inoculation.  The  color 
of  the  serum  should  be  pale  straw,  and  transparent.  In  addi- 
tion to  this,  serum  of  one-third  its  volume  of  nutrient  bouillon 
containing  10  per  cent  of  glucose  gives  a  medium  for  the  culti- 
vation of  diphtheria  bacillus. 

Inoculation.  —  This  is  the  term  employed  to  designate  that 
operation  adopted  by  the  experimenter  in  inserting  the  bacteria 
in  the  nutrient  media  for  the  purpose  of  causing  the  germs  to 


2l6  BIOLOGICAL   LABORATORY   METHODS 

grow  and  develop.    There  are  seven  methods  usually  in  practice 
among  bacteriologists  for  the  accomplishment  of  this  operation : 

1.  Tube  culture. 

2.  Dilution  method  (after  Nageli  and  Lister). 

3.  Fractional  culture  (employed  by  Koch). 

4.  Hanging-drop  culture  (after  Koch). 

5.  Plate  culture  (after  Koch).    - 

6.  Esmarch's  roll  culture. 

7.  Anaerobic  culture  (bacteria  excluded  from  air). 

Tube  culture.  —  The  test-tubes,  which  are*  employed  in  this 
method,  are  carefully  cleaned,  plugged  with  cotton,  and  sterilized. 
In  each  tube  is  placed  12  cc.  agar-agar  nutrient,  or  nutrient 
gelatin,  and  care  exercised  so  that  the  sides  of  the  tubes  are  not 
smeared  with  the  fluid.  The  tubes  are  then  placed  in  the  wire 
baskets  and  transferred  to  the  steam  sterilizer  for  fifteen  minutes 
each  day  for  three  days,  and  during  the  intervals  they  are  kept 
in  the  incubator  at  the  temperature  for  spore  germination.  If  at 
the  close  of  the  third  day  the  nutrient  fluids  come  from  the  in- 
cubator clear  and  without  spots,  it  can  be  assumed  that  all  life 
has  been  destroyed  in  the  tubes.  The  end  of  the  test-tube,  con- 
taining the  cotton  plug,  is  passed  through  the  flame  of  a  Bunsen 
burner  until  the  latter  ignites,  when  the  flame  is  put  out.  This 
action  is  for  the  purpose  of  destroying  all  bacteria  that  may  have 
lodged  in  the  dust  settling  from  the  air.  Now,  with  a  fine- 
pointed  pipette,  which  has  been  previously  carefully  sterilized, 
transfer  to  the  nutrient  in  the  tube  some  of  the  preparation  con- 
taining the  bacteria  which  is  to  be  subjected  to  examination. 
This  inoculation  is  accomplished  in  the  following  manner  :  Raise 
the  cotton  plug  slightly  and  insert  the  pipette  until  it  just  touches 
the  nutrient  agar,  and  then  press  it  slightly  below  the  surface 
of  the  nutrient  until  the  bacteria  in  the  pipette  find  lodgement  in 
the  fluid.  For  purposes  of  satisfactory  examination,  it  will  be 
well  to  place  the  bacteria  near  the  surface,  and  as  the  colonies 
grow  the  results  can  be  more  readily  determined  than  if  the 
inoculation  had  been  made  near  the  centre  of  the  nutrient  agar. 
If  the  test-tubes  were  somewhat  inclined  after  the  liquid  nutrient 


INOCULATION  2  I  / 

was  placed  in,  the  solidification  would  form  an  oblong  surface 
and  thus  expose  more  area  for  inoculation. 

Perform  this  work  of  inoculation  in  as  short  a  time  as  possible 
so  as  to  avoid  every  chance  of  contamination  from  the  germs  in 
the  air.  Place  the  inoculated  test-tubes  in  the  incubator  and 
keep  the  temperature  at  35°  C.  to  37°  C.  If  at  the  end  of  a  day 
or  so  the  agar  shows  a  turbid  condition,  or  spots  of  color  at 
places  near  the  surface,  the  experimenter  may  rest  satisfied  that 
his  work  is  progressing  well. 

Keep  careful  notes  of  the  following  items :  — 

1.  Source  of  bacteria  under  study. 

2.  Character  of  nutrient  used. 

3.  The  effects  on  the  nutrient. 

4.  Time  that  growth  began. 

5.  Temperature  sustained  in  the  incubator. 

6.  The  miscellaneous  appearance  of  the  colonies. 

7.  Make  drawings  of  all  stages  of  development. 

Dilution  method.  —  The  use  of  this  method  of  culture  is  to 
separate  the  bacteria  into  individuals,  or  at  least  into  small,  de- 
tached colonies  containing  a  small  number  of  individuals  of  the 
same  species.  This  work  is  done  for  the  purpose  of  facilitating 
the  study  of  the  special  disease,  and  to  determine  how  many 
species  of  bacteria  are  involved.  This  method  was  devised  by 
Nageli  and  Lister,  and  consists  in  diluting  with  sterilized  distilled 
water,  or  other  indifferent  fluid,  the  culture  fluid  containing  the 
bacteria,  and  then,  by  means  of  the  pipette,  inoculating  other 
tubes  which  have  sterilized  nutrient  agar  with  a  fraction  of  a 
drop  from  the  diluted  mass  of  bacteria.  Or  a  drop  is  drawn 
from  the  diluted  mass  and  the  pipette  containing  the  drop  is 
dipped  in  a  fresh  supply  of  sterilized  distilled  water,  or  well- 
boiled  saline  solution  (0.6  per  cent),  and  the  liquid  is  drawn  up 
into  the  pipette  so  that  the  dilution  of  the  drop  is  carried  still 
further,  one  thousand  fold  or  more,  and  from  this  dilution  other 
tubes  are  inoculated  with  the  smallest  fraction.  These  freshly 
inoculated  tubes  are  now  placed  in  the  incubator  and  the  cultures 
developed. 


2l8  BIOLOGICAL  LABORATORY   METHODS 

After  the  expiration  of  twenty-four  hours  an  examination  of 
the  tubes  is  made,  and  it  will  be  noted  that  some  have  clearly 
denned  spots  showing  bacteria  growths  of  probably  one  species, 
while  other  tubes  have  their  nutrient  agar  unchanged,  showing 
no  inoculation  from  the  portion  of  the  diluted  fluid  transmitted 
to  them  from  the  pipette.  Of  course  it  will  be  readily  seen  that 
this  dilution  may  be  carried  to  great  degree.  Instead  of  using 
the  pipette  to  accomplish  the  dilution,  a  drop  of  the  first  diluted 
mass  may  be  placed  in  a  liter  of  the  sterilized  distilled  water 
and  a  dilution  of  i  :  1,000,000  or  even  more  be  secured. 

Fractional  culture.  —  This  is  Koch's  method.  The  culture  is 
based  on  the  fact  that  one  species  of  bacteria  will  grow  readily 
under  certain  conditions  of  temperature  of  the  nutrient  fluid, 
while  another  species  will  not  thrive  so  well ;  the  first,  therefore, 
will  ultimately  crowd  out  the  latter  and  take  possession  of  the 
field.  The  steps  of  procedure  are  as  follows  :  — 

Inoculate  a  tube  with  sterilized  nutrient  fluid,  in  the  manner 
described  under  tube  culture,  and  place  it  in  the  incubator. 
After  the  colony  is  under  good  process  of  growth,  a  small  portion 
is  extracted  by  means  of  the  sterilized  pipette  and  transferred  to 
a  fresh  tube  of  sterilized  nutrient  fluid,  and  this  is  also  placed 
in  the  incubator,  where  a  constant  temperature  is  maintained  of 
35°  C.,  and  after  the  growth  begins,  a  third  tube  is  inoculated 
from  this  and  permitted  to  germinate  as  before,  and  a  fourth 
tube  is  inoculated  from  the  third,  and  so  on  until  the  reduction 
is  carried  so  far  as  to  insure  the  separation  of  the  species  ;  and 
it  is  claimed  that  in  this  manner  it  is  possible  to  obtain  pure 
cultures  of  individuals  of  the  same  species. 

Hanging-drop  culture.  —  This  was  also  devised  by  Koch,  and 
is  an  excellent  method  for  examining  the  growth  of  bacteria  in 
all  phases  under  the  microscope,  which  is  rather  difficult  of  ac- 
complishment with  the  other  methods  of  culture  without  extract- 
ing some  of  the  culture  and  mounting  it  on  a  glass  slip. 

Make  a  high  ring  with  glass  or  rubber  (Fig.  108)  on  a  slip  by 
cementing  to  the  slip  two  or  more  glass  rings  sold  by  any  of  the 
dealers  in  microscopical  material.  After  the  cement  well  hardens, 


PLATE  CULTURE  2IQ 

wash  in  a  solution  of  corrosive  sublimate  and  place  in  the  hot-air 
sterilizer.  Clean  in  the  same  manner  a  cover-glass  and  sterilize  ; 
place  in  the  centre  of  this  glass  a  drop  of  sterilized  nutrient 
fluid.  Inoculate  in  the  usual  way  and  put  the  cover-glass  on 
the  ring,  around  the  edges  of  which  has  been  smeared  sterilized 
vaseline.  Transfer  to  the  incubator  in  a  temperature  of  35°  C., 
and  after  the  growth  has  begun,  the  slide  can  be  placed  on  the 


FIG.  108.  —  Hanging-drop  Culture. 

stage  of  the  microscope  and  the  development  of  the  bacteria 
examined  at  pleasure  without  disturbing  the  colony. 

Plate  culture.  —  This  is  also  a  method  for  isolating  bacteria 
and  the  separation  of  species  into  distinct  colonies. 

A  tube  of  sterile  nutrient  fluid  is  warmed  until  the  fluid  be- 
comes liquid,  when  it  is  inoculated  with  a  trace  of  bacterial  mix- 
ture by  means  of  the  sterilized  pipette,  and  the  mass  is  well 
shaken  so  that  the  bacteria  are  well  distributed  throughout  the 
nutrient  agar  or  gelatin.  This  is  now  quickly  transferred  to  the 
bottom  of  a  Petri  dish  and  spread  out  over  the  surface.  The 
Petri  dish,  resting  on  a  glass  plate,  is  at  once  covered  by  another 
slightly  larger  dish,  and  over  these  dishes  is  placed  a  bell-glass 
jar,  around  the  edges  of  which  is  smeared  vaseline,  so  that  the 
contact  with  the  glass  plate  will  make  an  air-tight  connection. 
In  the  bell-glass  jar  is  also  placed  dampened  blotting-paper,  in 
order  that  the  air  maybe  kept  in  a  moist  condition.  Of  course, 
in  all  of  these  experiments  it  must  be  understood  that  all  dishes 
and  utensils  used  are  well  sterilized  before  the  inoculation  is 
performed.  The  nivellating  apparatus  illustrated  in  Fig.  109 
will  enable  the  experimenter  to  level  the  plate  culture,  and  thus 
preserve  a  uniform  depth  in  the  nutrient  fluid. 

If  the  nutrient  fluid  is  gelatin,  the  temperature  of  the  incuba- 
tor in  which  the  Petri  dishes  have  been  placed  must  be  kept  at 
a  temperature  near  20°  or  22°  C.  to  insure  the  solidity  of  the 


220 


BIOLOGICAL   LABORATORY   METHODS 


gelatin.  If  agar-agar  is  used  in  the  place  of  gelatin,  the  tem- 
perature can  be  raised  to  35°  C.  Experiments  have  proven  that 
some  bacteria  thrive  well  at  the  temperature  required  for  gelatin, 
while  they  would  be  destroyed  or  retarded  in  growth  if  placed 
in  agar  with  a  temperature  at  35°  C.  Other  forms  require 
higher  temperatures  to  germinate  and  produce  the  best  results,  so 
that  agar-agar  nutrient  must  be  the  fluid  used  for  the  experiment. 
If  a  still  further  differentiation  of  the  bacteria  is  desired,  a 

small  portion  of 
the  nutrient  fluid 
may  be  taken 
from  the  Petri 
dish  after  growth 
begins  and  a  new 
plate  culture  de- 
veloped. In  this 
manner  the  spe- 
cies may  be  sepa- 
rated into  colonies 
for  detailed  study. 
•Ex-march's  roll 

culture .  —  This 
FIG.  109.  -  Nivellating  Apparatus.  method  wag  exten_ 

sively  used  before  the  introduction  of  the  Petri  dish  method,  but 
it  has  now  been  almost  entirely  superseded  by  the  latter.  Like 
Koch's  plate  culture,  the  prime  object  of  the  roll  culture  is  for 
the  purpose  of  separating  the  bacteria  into  colonies  of  distinct 
species,  and  the  modus  operandi  consists  in  liquefying  the  fluid 
nutrient  gelatin  in  a  large  tube,  inoculating,  and  revolving  the 
tube  inclined  on  a  block  of  ice  until  the  gelatin  solidifies  on  the 
sides  of  the  tube.  Care  must  be  taken  not  to  permit  the  gelatin 
to  come  in  contact  with  the  cotton  plug.  It  is  best  to  use  gelatin 
with  this  method,  because  the  agar  does  not  adhere  so  well  to 
the  sides  of  the  glass  tube. 

Anaerobic  culture.  —  Some  bacteria  do  not  grow  freely  in  oxy- 
gen, and  in  the  study  of  such  forms  it  becomes  necessary  to 


ANAEROBIC  CULTURE 


221 


substitute  a  gas  for  the  oxygen  or  air,  or  to  cultivate  the  bacilli 
in  bouillon  covered  with  a  substance  impervious  to  air.  There 
are  several  devices  proposed  by  workers  in  bacteriology  for  ac- 
complishing this  exclusion  of  the  oxygen  of  the  air.  One  of  the 
most  simple  and  effective  is  that  suggested  by  Dr.  Theodore 
Kasparec  of  Vienna  : 1  — 

"  In  order  to  be  able  to  cultivate  tetanus  bacilli  in  bouillon  in 
a  simple  manner,  I  prepared  the  cultures  in  ordinary  flasks  and 


FIG.  no.  FIG.  in. 

Kasparec's  Anaerobic  Culture  Apparatus. 

selected  the  paraffin  method,  as  recommended  by  Kitasato,  for 
excluding  the  air.  As  pouring  the  liquid  paraffin  upon  the  in- 
oculated bouillon  did  not  appear  practical  enough,  on  account 
of  the  danger  of  foreign  germs  entering  while  pouring,  the 
smearing  of  the  neck  of  the  glass  with  paraffin,  as  well  as  the 
influence  of  the  heat  of  the  melted  paraffin,  I  had  a  special  flask 
made  for  the  purpose,  which  had  proved  very  reliable  in  keeping 
the  culture  air-tight. 

"  The  flask  intended  for  the  purpose  may  be  a  spherical  one, 
may  be  of  any  desired  size,  with  a  rather  long  neck  tapering 
toward  the  top.  A  small  tube  terminating  in  a  bulb  is  blown 

1  Central-Blatt  f.  Bakt.,  Bd.  XX,  p.  536,  and  Jour.  App.  Mic.,  Vol.  I,  p.  34. 


222 


BIOLOGICAL   LABORATORY   METHODS 


into  the  side  of  the  neck  of  the  flask  about  one  centimeter  from 
C  (Fig.  no).  The  flask  is  first  filled  with  bouillon  almost  to  the 
neck,  and  about  three  cubic  centimeters  of  liquid  paraffin  are  then 
added,  after  which  the  whole  is  sterilized  in  the  steam  sterilizer. 
The  heat  expands  the  bouillon,  causing  the  paraffin  to  rise  in  the 
neck  of  the  flask  and  overflow  into  the  side  neck  and  small  bulb 
A  (Fig.  in),  so  that  after  sterilization  there  is  only  a  very  thin 
film  of  paraffin  remaining  on  the  top  of  the  culture  medium  at  C. 
"  During  the  heating  a  large  portion  of  the  air  absorbed  by  the 
bouillon  is  driven  out,  and  its  reabsorption  while  the  flask  is 
cooling  is  prevented  by  the  thin  film  of  paraffin.  After  cooling, 
the  paraffin  hardens  into  a  solid  coating,  which  can  be  readily 
pierced  when  it  is  desired  to  inoculate  the  bouillon.  After  in- 
oculation the  hardened  paraffin  in  the  small  bulb  is  liquefied  by 
heating  slightly,  when  it  may  be  poured  upon  the  film  already 
formed  above  the  bouillon  by  slightly  inclining  the  flask.  Upon 
hardening  this  additional  paraffin  constitutes  an  almost  perfectly 

air-tight  layer,  which  becomes  even 
more  effective  by  being  pressed 
into  the  tapering  neck  when  the 
culture  is  heated  in  the  incubator. 
When  pouring  out  the  cultures  after 
growth  has  taken  place,  the  paraffin 
is  again  led  into  the  lateral  bulb, 
after  warming  the  vessel,  by  inclin- 
ing the  flask."1 

Another  simple  device,  suggested 
by  Turro  in  Cent./.  Bac.,  I,  31,  175, 
is  to  use  a  Petri  dish  with  a  glass 
circular  plate  resting  on  glass  sup- 
ports extending  from  the  bottom  to 
within  a  half-inch  of  the  top  of  the 
dish.  The  gelatin  culture  medium  is  spread  over  the  circular 
glass  top  and  inoculated,  and  then  inverted  to  its  position  on 

1  For  a  full  discussion  of  the  methods  of  cultivating  anaerobic  bacteria,  see 
Jour.  App.  Mic.,  Vol.  V,  p.  1800. 


ANAEROBIC   CULTURE 


223 


the  glass  supports  in  the  Petri  dish.  Before  this  top  is  placed 
on,  however,  there  is  poured  into  the  dish  a  quantity  of  pyro- 
gallic acid  and  NaOH,  and  then  the  top  is  sealed  down  with 
melted  paraffin,  which  will  prevent  the  entrance  of  air.  The 
pyrogallic  acid  will  soon  absorb  the  oxygen 
remaining  in  the  dish,  and  it  will  then  be 
possible  for  the  bacteria  to  grow  free  from 
the  oxygen. 

Novy  has  devised  an  apparatus  of  tube 
and  plate  cultures  by  the  gas  or  pyrogallate 
methods  (Figs.  112  and  113),  which  pos- 
sesses some  advantages  worthy  of  con- 
sideration. This  instrument,  as  shown  in 
Fig.  119,  is  a  glass  vessel  made  in  two  sec- 
tions, so  that  Petri  dishes  as  well  as  tubes  FIG.  113.— Novy's  Cui- 

Can  be   placed   in    it.      The   top    Section    is    ture  Apparatus  for  Tubes. 

clamped  on  and  made  air-tight,  and  the  hydrogen  gas  passed 
through  until  the  air  is  driven  out.  In  the  pyrogallate  method, 
3  to  5  grams  of  pyrogallic  acid  are  placed  in  the  bottom  of  the 
apparatus  in  a  dish  and  the  Petri  dishes  placed  on  top  of  it,  with 
a  narrow  strip  of  glass  intervening.  The  apparatus  is  now 
closed,  excepting  a  narrow  slit,  through  which  is  introduced  as 
quickly  as  possible  25  cc.  of  concentrated  potash  solution  (1:4). 

The  top  is  then 
securely  fastened 
down.  (See  Jour. 
App.  Mic.,  Vol.  II, 
p.  267.) 

Wolffhuegel's 
counting  apparatus. 

FIG.  114.  —  Wolff  huegel  s  Counting  Apparatus. 

—  It  now  becomes 

necessary,  in  the  culture  of  bacteria,  to  know  how  many  individ- 
uals belong  to  the  colony,  and  in  order  to  do  so  with  any  degree 
of  accuracy  a  counting  apparatus  must  be  used.  The  above  is 
one  of  the  best  (Fig.  114),  and  is  well  known  among  microscopists. 
The  rulings  on  the  plate  are  i  centimeter  square,  and  there  are 


224  BIOLOGICAL  LABORATORY   METHODS 

diagonal  rows  of  these  squares  ruled  to  0.3  centimeter  squares. 
There  are  two  background  plates,  one  white  and  the  other  black, 
so  as  to  facilitate  the  reading  of  the  measures  on  the  instru- 
ment. The  culture  plate  or  the  Petri  dish  is  placed  on  this 
instrument,  and  the  individual  bacteria  are  counted  which  cover 
one  of  the  graduated  squares  and  an  estimate  made  from  this  for 
the  entire  colony. 


CHAPTER   XV 

BLEACHING,    DECALCIFICATION,    INJECTION,    MACERATION 

Bleaching.  —  Some  of  the  objects  subjected  to  microscopical 
examination  are  so  opaque  with  pigments  and  other  matters,  it 
becomes  necessary  to  bleach  or  clarify  them  with  some  chemical 
which  will  act  on  the  pigment  and  dissolve  it  out  of  the  speci- 
men. The  chemicals  generally  suitable  for  this  purpose  are  so 
active  in  their  attack  on  the  subject  to  be  eliminated  that,  unless 
great  care  is  exercised,  the  delicate  tissues  will  be  seriously 
injured.  Of  course  it  will  be  readily  understood  that  in  the 
case  of  those  experiments  where  there  are  soft  cell  contents,  or 
the  fine  markings  on  the  delicate  cell  walls,  then  the  strong 
alkalies  and  acids  used  in  bleaching  must  not  be  employed. 

It  becomes  necessary  also  to  bleach  when  specimens  have 
been  overstained  in  such  stains  as  chloride  of  gold  and  nitrate 
of  silver  or  fixed  in  osmic  acid  solutions.  When  chlorid  of 
gold,  which  has  been  used  in  staining  nerve  tissues,  has  over- 
stained  the  section,  the  extra  color  may  be  extracted  by  using 
cyanide  of  potassium.  This  chemical  may  also  be  used  in 
treating  sections  overstained  with  nitrate  of  silver.  The  cyanide 
of  potassium  should  be  dissolved  in  water  at  the  rate  of  i  :  10. 

Insects  can  be  bleached  in  a  solution  of  peroxid  of  hydrogen, 
so  that  the  respiratory  organs  may  be  clearly  and  satisfactorily 
examined  under  the  microscope. 

A  weak  water  solution  of  potassium  hydroxid  is  a  bleaching 
agent  which  acts  on  protoplasm  and  transforms  it  into  a  clear 
mass.  It  also  acts  on  the  cell  walls.  Hanstein  used  it  in  his 
experiments  on  merismotic  tissue  and  germ  development.  After 
treatment  in  the  potassium  hydroxid,  wash  and  soak  in  glycerin 
diluted  in  water  or  alcohol,  when  the  specimen  will  become 

225 


226  BIOLOGICAL  LABORATORY   METHODS 

clear  and  so  transparent  that  the  cell  walls  almost  disappear, 
but  by  treating  with  weak  solution  of  alum  the  walls  reappear. 
Resins  and  fat  masses  will  yield  to  the  action  of  this  bleaching 
agent  by  soaking  for  a  short  time  in  the  moderately  strong  solu- 
tion of  the  chemical  and  then  washing  with  strong  alcohol.  If 
the  tissue  shrinks,  because  of  the  too  rapid  action  of  the  gums 
and  the  strong  effects  of  the  alcohol,  the  cells  can  be  made  to 
retain  their  normal  shapes  by  imperfectly  washing  with  the 
alcohol  and  the  repeated  application  of  water.  Transfer  to 
water  containing  a  little  hydrochloric  acid. 

Bleaching  animals  and  sections  fixed  with  osmic  acid  solutions 
(Dr.  D.  Carazzi,  Zool.  Anzeig.,  XVII,  p.  135,  1894).  Peroxid 
of  sodium  is  used  (Na2O2).  When  dissolved  in  water  the  yellow 
powder  becomes  alkaline  and  forms  caustic  soda.  Mixed  with 
tartaric  acid  or  acetic  acid  the  soda  combines  with  the  acid.  A 
10  per  cent  solution  of  the  acid  is  put  in  a  vessel,  and  a  small 
quantity  of  the  peroxid  of  soda  added  ;  then  70  per  cent  alcohol  is 
slowly  dropped  on  the  surface  of  the  solution.  Place  the  object  in 
the  alcohol  thus  added  to  the  peroxid  of  soda,  and  as  the  oxy- 
gen escapes  from  the  water  and  rise^  in  the  alcohol  it  is  slowly 
dissolved  and  the  specimen  is  bleached. 

Marsh's  method  for  bleaching.  —  This  consists  in  the  applica- 
tion of  free  chlorin  to  the  specimen.  Two  wide-mouth  bottles, 
A  and  B  (Fig.  115),  are  provided  with  securely  fitting  corks.  In 
A  is  placed  potassium  chlorate  and  hydrochloric  acid,  and  in  B 
is  water  with  the  specimen  to  be  bleached.  Connect  the  two 
bottles  with  a  glass  tube  C,  so  that  the  end  which  enters  the 
bottle  A  will  go  just  below  the  under  surface  of  the  cork,  and 
the  other  end  in  B  must  extend  to  the  bottom  of  the  bottle. 
The  chlorin  gas  evolved  by  the  action  of  the  hydrochloric  acid 
on  the  potassium  chlorate  will  pass  over  into  the  bottle  B  and 
bleach  the  specimens,  and  the  extra  gas  will  escape  through  a 
notch  in  the  cork  at  D.  Wash  in  running  water  after  bleaching 
until  all  of  the  chlorin  is  taken  out  of  the  specimens. 

Mayer's  chlorin  method  differs  from  Marsh's  in  the  way  the 
crystals  of  chlorate  of  potash  are  used.  The  vessel  containing 


BLEACHING 


227 


the  chlorate  of  potash  and  the  hydrochloric  acid  also  contains 
the  specimens  to  be  bleached.  As  soon  as  the  chlorin  gas 
begins  to  evolve,  a  few  cubic  centimeters  of  50  to  70  per  cent 
alcohol  are  added,  and  the  specimens,  previously  soaked  in  90 
per  cent  alcohol,  are  placed  in  the  alcohol  floating  on  the 
chlorate  of  potash  solution.  They  soon  sink  to  the  bottom, 


FIG.  115.  —  Marsh's  Apparatus  for  Bleaching. 

and  the  bleaching  will  be  completed  in  a  few  hours.  This 
method  will  correct  the  staining  due  to  osmic  acid,  and  the 
chlorin  gas  will  also  eliminate  natural  colors  in  the  sections. 
The  action  is  aided  by  placing  the  apparatus  in  the  water-bath 
under  a  gentle  heat.  After  bleaching  remove  the  specimens  to 
pure  alcohol. 

Decalcification.  —  In  order  to  secure  satisfactory  sections  of 
bones,  teeth,  and  other  specimens  containing  calcium,  it  becomes 
necessary  first  to  extract  all  the  lime  salts.  The  agents  gener- 
ally used  for  this  purpose  are  hydrochloric  and  nitric  acids. 


228  BIOLOGICAL  LABORATORY  METHODS 

The  acids  must  be  in  a  diluted  form,  and  since  in  the  case  of 
the  hydrochloric  acid  swellings  of  the  tissue  result,  chromic  acid 
or  alcohol  must  be  combined  with  it  to  prevent  in  a  measure 
this  trouble.  A  solution  of  common  salt,  10  to  15  per  cent, 
added  to  a  3  per  cent  solution  of  hydrochloric  acid  will  also 
prevent  the  swelling.  Nitric  acid  does  not  produce  this  trouble. 
Before  decalcification  the  specimen  must  be  well  fixed  in 
alcohol  or  in  picric  acid,  or  in  Tellyesmiczky's  acetic  bichromic 
solution,  viz. :  — 

Bichromate         .         .         ...         ,         .         .         3  grammes 
Glacial  acetic  acid      .         .         «...         .         .         5  cc. 
.    Water         .        .       '*.        ...        ...     100  cc. 

The  specimens  must  be  treated  in  large  quantities  of  the 
decalcifying  solution  with  frequent  changing  to  fresh  quantities. 
After  the  decalcification  is  completed  wash  in  running  water, 
or  soak  for  several  days.  Now  transfer  to  several  strengths 
of  alcohol  to  harden  and  dehydrate. 

Haug's  decalcifying  solution.  —  This  is  one  of  the  most  rapid 
decalcifying  agents  known  to  microscopists.  The  addition  of 
the  phloroglucin  is  to  reduce  the  injurious  effects  of  the  strong 
nitric  acid.  Bones  placed  in  this  solution  will  become  decalci- 
fied in  a  few  hours,  but  it  is  best  to  watch  carefully  the  action 
of  the  solution  because  of  its  very  rapid  work.  Wash  several 
days  in  running  water. 

Phloroglucin       .         .         .         .         .         ...         I  gramme 

Nitric  acid  (pure)       .        .        »     .   .'       ..        .        .       10  cc. 

Warm  slowly  and  add  100  cc.  of  water  and  10  cc.  nitric  acid. 
After  washing  the  tissues  pass  them  through  70  per  cent  and 
95  per  cent  alcohol. 

Dr.  E.  Rousseau  has  devised  the  following  method  for  decal- 
cifying :  — 

"  The  objects  to  be  decalcified,  which  should  not  be  too  large, 
are  imbedded  in  celloidin  in  the  ordinary  way,  i.e.  after  fixation, 
are  dehydrated  in  alcohol,  and  then,  after  having  been  immersed 
in  a  mixture  of  equal  parts  of  ether  and  alcohol,  are  impreg- 


INJECTION  229 

nated  with  celloidin  in  solutions  of  increasing  strength  (4  per 
cent,  8  per  cent,  12  per  cent).  When  the  objects  are  suffi- 
ciently saturated  with  celloidin,  the  latter  is  hardened  by  slow 
evaporation,  by  alcohol,  or  by  chloroform.  The  celloidin  blocks 
containing  the  objects  are  next  immersed  in  a  mixture  of  alcohol 
at  90°  and  nitric  acid,  the  proportion  of  the  latter  being  regu- 
lated by  the  amount  of  calcareous  matter  to  be  got  rid  of.  The 
author  uses  10  to  50  parts  of  nitric  acid  to  100  of  alcohol. 
The  decalcifying  fluid  should  be  renewed  from  time  to  time, 
and  when  decalcification  is  complete,  the  excess  of  acid  is 
removed  by  washing  in  water  and  immersion  in  90  per  cent 
alcohol,  frequently  changed  for  several  days.  When  all  the 
acid  has  been  removed  the  celloidin  block  may  be  sectioned. 
Now  it  may  happen  that,  owing  to  extensive  decalcification, 
there  will  be  large  gaps  in  the  objects  which  would  be  a  source 
of  considerable  inconvenience  in  making  and  manipulating  the 
sections.  The  difficulty  is,  however,  easily  overcome  by  filling 
up  the  holes  with  4  per  cent  celloidin,  and  allowing  this  to  set 
by  evaporation.  This  method  possesses  many  advantages  — 
there  is  no  deformity  of  the  animals  or  tissues,  which  are 
observed  in  situ,  and  it  is  easy  and  rapid."  (Jour.  Roy.  Mic. 
Soc.,  p.  375,  1898.) 

Injection.  —  In  normal  histology  it  is  a  common  practice  to  fill 
the  vascular  system  with  coloring  matter  when  it  is  desired  to 
study  the  arterioles  and  capillaries  of  animals.  The  method  of 
injecting  the  -coloring  matter  into  the  arteries  and  veins  of  the 
animal  is  difficult,  and  success  can  only  be  secured  by  long  prac- 
tice. The  results,  however,  are  exceedingly  beautiful,  and,  in 
some  cases,  the  injection  is  necessary  in  order  to  show  the  rela- 
tion of  the  capillaries  to  the  other  elements  of  the  organ  under 
examination.  The  student  should  not  become  discouraged  if  his 
first  attempts  are  not  successful,  but  he  must  have  patience  and 
persevere  if  proficiency  in  the  work  is  to  be  attained. 

The  coloring  matter  is  held  in  suspension  in  either  glycerin  or 
gelatin,  and,  the  character  of  the  mass  depends  on  the  kind  of 
color  to  be  injected.  The  glycerin  and  gelatin  are  termed  the 


230  BIOLOGICAL  LABORATORY  METHODS 

vehicles,  and  the  combination  of  the  vehicle  with  the  color  is 
called  the  injectmi  mass.  Glycerin  is  preferable  to  gelatin  be- 
cause of  its  convenience  and  the  good  results  secured,  and 
because  the  mass  can  be  used  cold.  Glycerin  masses,  however, 
have  defects,  as  stated  by  Lee  in  his  "  Vade-Mecum,"  "  for  in- 
jection of  fresh  specimens  —  that  is,  those  in  which  rigor  mortis 
has  not  set  in ;  they  stimulate  the  contraction  of  the  arteries. 
In  these  cases  it  may  be  advisable  to  use  nitrite  of  amyl  as 
a  vasodilator.  The  animal  may  be  anaesthetized  with  a  mix- 
ture of  ether  and  nitrite  of  amyl,  and  finally  killed  with  pure 
nitrite." 

To  inject  with  gelatin  some  difficulty  is  experienced  because 
of  the  necessity  of  keeping  the  mass  and  the  instruments  and 
the  object  in  a  warm  condition.  There  are  two  gelatin  masses 
which  are  generally  used  for  injecting,  viz. :  Fol's  carmine  and 
gelatin  mass,  and  Thiersch's  Prussian  blue  gelatin  mass. 

Good  photographic  gelatin  is  soaked  for  several  hours  in  a 
small  quantity  of  water,  the  water  is  then  poured  off  and  the 
mass  warmed  over  the  water-bath  until  the  gelatin  melts.  Car- 
mine is  added  to  strong  ammonia,  diluted  with  three  parts  of 
water  until  saturation,  and  the  solution  is  poured  into  the  gelatin 
while  stirring.  The  correct  proportions  are  :  — 

Gelatin I  kilogram 

Water    .         ,,         .         .         .         .         ,         .         .         small  quantity 
Carmine-ammonia          .         .         .         ...         I  liter 

To  prevent  the  mass  from  becoming  opaque  by  the  precipita- 
tion of  the  carmine,  it  must  be  neutralized  by  the  addition  of 
acetic  acid  until  the  color  is  changed  from  a  dark  purple  to  a 
blood-red.  After  congealing,  the  gelatin  mixture  is  cut  up  and 
washed  in  a  sieve  for  several  hours  to  dissolve  out  the  ammonia 
and  acetic  acid.  It  is  then  dried  on  paper  which  has  been  pre- 
viously covered  with  paraffin.  For  use  melt  in  the  water-bath 
and  inject  in  a  warm  condition. 

Thiersch's  Prussian  blue  gelatin  mass.  —  Make  saturated  solu- 
tions of  (i)  sulphate  of  iron,  (2)  oxalic  acid,  (3)  red  prussiate  of 


INJECTION  231 

potash,  (4)  and  prepare  a  warm  solution  of  i  part  of  gelatin  in  2 
parts  of  water.  (5)  Mix  in  a  separate  vessel  12  cc.  of  solution 
(i)  with  30  grammes  of  solution  (4)  at  a  temperature  of  25°  C. 
(6)  In  another  dish  mix  60  grammes  of  solution  (4)  with  24  cc. 
of  (3),  to  which  add  24  cc.  of  solution  (2),  and  stir  thoroughly. 
To  this  last,  (6),  slowly  add  solution  (5)  and  stir,  with  the  tem- 
perature at  25°  C.,  until  the  Prussian  blue  is  precipitated.  Then 
heat  over  water-bath  at  a  temperature  of  70°  C.  and  filter  through 
flannel. 

Beale's  Prussian  blue  glycerin  mass  is  probably  the  best  of 
the  glycerin  masses  for  students'  work,  and  the  formula  is  as 
follows :  — 

Price's  glycerin 60  cc. 

Ferrocyanide  of  potassium 2  decigrams 

Tincture  sesquichlorid  of  iron         .         .         .         .10  drops 
Concentrated  hydrochloric  acid      .                  .         .  3  drops 

Water 30  cc. 

Dissolve  the  ferrocyanide  of  potassium  in  30  cc.  of  glycerin, 
and  the  sesquichloride  of  iron  in  30  cc.  of  glycerin,  and  add  drop 
by  drop  to  the  ferrocyanide  of  potassium  solution.  Add  the 
water  and  then  the  hydrochloric  acid. 

Starch  injection.  —  Ad  Pausch,1  in  1877,  first  introduced  the 
method  of  using  starch  as  an  injection  substance,  and  afterward 
his  method  was  modified  by  Browning,  Delia  Rossa,  Gage, 
Meyer,  and  Wikssemski.  Pausch  first  recommended  wheat 
flour,  but  later  experiments  proved  that  pure  starch  was  superior 
to  flour.  Starch  is  insoluble  in  alcohol  and  cold  water,  and 
when  it  is  injected  in  the  animal  it  becomes  hard  simply  by 
exudation  of  the  liquid  containing  it.  It  may  be  forced  up  to 
the  capillaries,  and,  "  hardening  rapidly,  it  leaves  the  vessels 
flexible."  The  softness  of  the  starch  and  its  freedom  from  all 
gritty  material  permits  the  cutting  of  sections  without  injury  to 
the  microtome  knife. 

1  Arch.  f.  Anat.  Med.  Entwick.,  p.  480,  1877.  Amer.  Nat.,  XVIII,  p.  958,  1884. 
Annals  Anat.  and  Surg.,  p.  24,  1884.  Jour.  Roy.  Mic.  Soc.,  p.  979,  1884  ;  pp.  105-106, 
1888.  Amer.  Man.  Mic.  Jour.,  IX,  p.  195,  1888. 


232  BIOLOGICAL  LABORATORY   METHODS 

MASS  FOR  ORDINARY  INJECTION  WITH  STARCH  :  — 

Dry  starch 100  vols. 

Aqueous  solution  of  chloral  hydrate  2\  per  cent  .         .  100  vols. 

Alcohol  95  per  cent \  vol. 

Coloring  material \  vol. 

Filter  through  several  thicknesses  of  cambric  and  stir  on  the 
filter  to  prevent  settling. 

The  addition  of  the  alcohol  and  the  chloral  prevents  fermenta- 
tion, and  when  the  mass  is  injected  into  the  animal  the  alcohol 
gives  greater  fluidity  to  the  mass  and  rapidly  increases  the  hard- 
ening properties  after  the  starch  reaches  the  vessels  in  the  system 
under  study. 

Among  the  colors  of  most  importance  for  this  injection  mass 
are  red  lead,  vermilion,  chrome-orange,  and  ultramarine. 

PREPARATION  OF  THE  COLOR  :  — 

Dry  color       .         .         ...         .         ,         .         .         100  vols. 

Glycerin         .         .         .         ...         .         .         .         .         100  vols. 

Alcohol  95  per  cent       .         .         .     '   v-      *       '.         .         100  vols. 

Grind  the  color  with  the  liquid  in  a  mortar  to  prevent  the 
formation  of  lumps  in  the  mass.  Keep  in  a  bottle,  and  shake 
before  using. 

Special  injection  masses.  —  For  brains  and  other  rapidly  per- 
ishing organs : — 

Starch -    -  .  .  .  100  vols. 

Chloral  hydrate  5  per  cent  aqueous  solution  *  .  .  50  vols. 

Color  mixture        .         .         .         .         .        \  .  ^  .  75  vols. 

Alcohol  95  per  cent       .         .         .         «         .  .  .  25  vols. 

To  inject  fine  vessels,  first  use  a  diluted  form  of  the  stock  so- 
lution with  equal  volumes  of  water,  or  of  the  chloral  hydrate, 
and  then  follow  with  the  stronger  solution.  Perform  the  injec- 
tion quickly,  because  the  exudation  takes  place  so  soon  that  the 
mass  hardens  rapidly.  In  the  case  of  very  large  vessels,  like 
the  aorta,  increase  the  amount  of  the  starch. 

Other  injection  masses.  —  Some  authorities  have  recommended 
the  use  of  plaster  of  Paris,  wax,  asphaltum,  shellac,  india  ink, 


INJECTION  233 

and  gum  arable  and  other  substances,  but  Lee  and  some  other 
authorities  have  claimed  that  "  all  formulae  which  only  give 
opaque  masses,  or  are  only  suitable  for  coarse  injections  for 
naked-eye  study,  have  been  suppressed." 

Double  injection  with  gelatin  and  plaster  of  Paris  may  be  ac- 
complished by  forcing  in  the  animal,  first,  colored  solution  of 
gelatin,  and  followed  by  a  differently  colored  plaster  of  Paris. 
The  latter  goes  only  as  far  as  the  capillaries,  and  we  thus  have 
the  arteries  and  larger  vessels  stained  with  the  plaster  of  Paris 
and  the  capillaries  stained  with  the  gelatin. 

The  Apparatus  required  for  Injection    Work 

1.  Several  sizes  of  cannulas  and  stopcocks,  fitted  to  a  syr- 
inge. Or  a  constant-pressure  apparatus. 

2.  Needles  with  curved  points. 

3.  No.  10  Chinese  silk. 

4.  Strong  twine. 

5.  Sharp  knife  or  scalpel. 

6.  Dissecting  pans,  or  similar  vessels. 

7.  Kettle  for  hot  water. 

8.  Vessel  for  ice-water. 

9.  Box,  with  a  close-fitting  top,  hinged,  in  which  to  place  the 
animal  for  killing  with  chloroform  or  ether. 

10.  Bottle  of  chloroform  or  ether. 

11.  Forceps. 

12.  Injection  masses  in  large  bottles. 

There  are  three  methods  in  practice  for  injecting  fluids  into 
animals :  — 

1 .  By  means  of  the  syringe  with  pressure  from  the  hands  of 
the  operator. 

2.  The  mechanical  or  constant-pressure  injection. 

3.  The  natural  injection,  or  by  introducing  pigments   into 
the  circulating  system  of  the  animal. 

By  the  first  system  the  syringe  should  be  warmed  by  filling 
several  times  with  warm  water  before  using  the  gelatin  mass. 
The  syringe  should  be  carefully  filled,  so  that  no  air  will  enter 


234 


BIOLOGICAL  LABORATORY  METHODS 


the  tissue  to  be  injected,  because  air  will  spoil  the  results.  Some 
practice  is  required  to  know  just  how  much  pressure  to  apply  to 
the  piston  of  the  syringe.  The  connections  must  all  fit  air-tight, 
which  may  be  determined  by  pressing  the  piston  to  its  lowest 
position  and  then,  with  the  fingers  over  the  end  of  the  nozzle, 


FlG.  116. —  Injection  Syringe. 

drawing  the  piston  out  and  letting  it  fly  back.  If  air-tight,  it 
will  return  quickly  to  its  former  position.  It  is  best  also  to  have 
the  rod  of  the  piston  graduated,  so  that  the  eye  may  determine 
whether  the  injection  mass  is  flowing  into  the  system. 

Two  or  three  different  sizes  of 
these  syringes  (Figs.  116  and  117) 
will  be  found  convenient  to  use 
with  small  and  large  animals. 
The  syringes  should  hold  about 
30  cc.  and  60  cc.  The  cannulae 
should  be  1.6  mm.,  0.8  mm.,  0.4 
mm.  in  diameter,  and  there  should 
be  on  each  projections,  or  arms, 
on  which  to  tie  them  to  the  vessels 
to  be  injected  (Fig.  118). 

The  Stroschein  injection  syr- 
inge is  made  of  glass,  and  it  is 
valuable  for  bacteriological  work 
because  it  is  easily  cleaned  and 
sterilized.  The  instrument  consists  of  three  pieces,  —  a  tube  A 
sliding  over  a  smaller  tube  B  by  means  of  a  rubber  connection, 
and  a  cannula  C.  The  apparatus  is  placed  in  operation  by  slip- 


FlG.  117.  —  Injection  Syringe. 


INJECTION 


235 


ping  A  as  far  down  on  B  as  possible  and  then  inserting  the 
end  of  the  cannula  in  the  injecting  fluid.  A  is  now  returned 
to  its  farthest  position  on  B,  and  the  suction  will  cause  the 


A 

B 

,(T 

' 

' 

•hi 

•I'N'hi  ^ 

-^ 

FlG.  118.  —  Stroschein's  Injection  Syringe. 

liquid  to  rise  in  B,  the  air  escaping  from  B  into  A  by  means 
of  the  orifice  O.  By  a  reverse  action  of  A  the  liquid  will  be 
expelled  from  the  cannula  into  the  tissues. 

The  second  method  is  applied  by  means  of  the  following  appara- 
tus, which  gives  a  constant  and  steady  flow  of  the  mass  into  the 
system,  and,  when  put  into  operation,  it  will  work  automatically. 


FlG.  119.  —  Sterling's  Constant  Pressure  Apparatus. 

Sterling's   constant  pressure  apparatus.  —  This  consists  of  a 
large,  wide-mouth  bottle  A  (Fig.  119),  which  has,  entering  a 


236        BIOLOGICAL  LABORATORY  METHODS 

closely  fitting  cork,  three  tubes,  C,  D,  E.  The  tube  D  extends 
to  the  bottom  of  the  bottle,  and  the  other  end  connects  with 
the  water  by  means  of  a  faucet.  C  connects  with  a  mercurial 
manometer  M,  and  E  extends  to  another  bottle  B,  containing  the 
injection  mass.  From  the  bottle  B  another  tube  ^extends  from 
the  "bottom  to  the  cannula  and  the  tissues  to  be  injected.  The 
operation  of  this  apparatus  is  as  follows  :  The  stopcock  on  D 
entering  the  water-main  is  opened,  and  the  water  flowing  into 
the  larger  bottle  causes  the  air  to  press  against  the  injection 
mass  in  the  bottle  B  and  force  it  into  the  tissues  through  the 
cannula  at  the  other  end  of  the  tube  E.  The  force  of  the  press- 
ure can  be  regulated  by  opening  the  stopcock  in  C  and  watching 
the  effects  on  the  mercury  in  the  bent  tube  on  the  manometer. 
If  the  flow  is  too  great,  the  stopcock  connecting  with  the  water- 
main  must  be  reduced.  In  this  manner  a  constant  and  regular 
flow  of  the  mass  into  the  system  can  be  maintained. 

Killing  the  animal.  —  Place  in  the  box  mentioned  above  the 
animal  to  be  treated  to  the  injection  mass  and  drop  in  a  wad  of 
cotton  saturated  with  chloroform,  close  down  the  top,  and  in 
about  fifteen  minutes  the  animal  will  be  dead.  A  rabbit 
is  probably  the  best  animal  on  which  the  student  may  experi- 
ment with  the  chance  of  ultimate  success.  As  soon  as  dead 
make  an  incision  through  the  thorax  by  cutting  the  sternal  ribs 
of  both  sides  "  sufficiently  far  from  the  middle  line  not  to  injure 
the  mammary  arteries,  cutting  across  the  posterior  end  of  the 
sternum  and  turning  it  forward.  Slit  open  the  pericardium 
and  make  a  large  incision,  by  a  single  cut  of  the  scissors,  in 
each  ventricle.  All  this  should  be  done  rapidly,  as  it  is  desirable 
to  get  rid  of  as  much  blood  as  possible.  Pass  a  ligature  round 
the  aorta  close  to  its  exit  from  the  heart,  and  give  it  a  single 
loose  tie.  When  the  bleeding  has  ceased,  sponge  the  blood 
from  the  heart,  and  pick  out  any  clots  which  may  have  formed 
in  the  left  ventricle,  pass  a  cannula  through  the  incision  in  the 
left  ventricle  into  the  aorta,  tighten  the  ligature,  and  knot 
it  firmly.  By  this  operation  the  whole  systemic  arteries  are 
injected.  The  pulmonary  arteries  may  be  filled  by  proceeding 


INJECTION  237 

similarly  on  the  right  side.  The  portal  vein  is  readily  injected 
from  its  branch  to  the  caudate  lobe,  the  cannula  being  directed 
toward  the  main  trunk.  The  injection  of  the  systemic  veins 
is  more  difficult.  The  precavals  may  be  filled  from  the  external 
jugular,  the  post-caval  from  the  external  iliac,  the  cannula  in  both 
cases  being  directed  toward  the  heart."  (Jour.  Mic.  Nat.  Sci) 
The  work  of  injection  is  completed  as  soon  as  the  extremities 
of  the  animal  begin  to  show  a  decided  coloration  of  the  injected 
mass.  Then  tie  all  open  vessels  and  place  the  animal  in  cold 
water.  In  an  hour  or  so  the  tissues  may  be  dissected  and 
transferred  to  alcohol  to  fix  and  dehydrate. 

Maceration.  —  The  object  of  this  treatment  is  to  separate  the 
tissues  from  each  other  by  chemicals  instead  of  dissecting  with 
sharp-pointed  needles,  the  chemicals  acting  on  the  substances 
which  cement  the  tissues  together  and  dissolving  them  so  that 
the  tissues  easily  fall  apart.  The  following  are  some  of  the  sub- 
stances used  for  this  purpose  :  — 

Alcohol,  90  per  cent  in  2  parts  of  water. 

Sodium  chloride. 

Caustic  soda. 

Nitric  acid. 

Osmic  acid. 

Acetic  acid. 

Permanganate  of  potash. 

Formaldehyde. 

Landois's  solution,  viz. :  — 

Saturated  solution  of  neutral  chromate  of  ammonia         .  5  parts 

Saturated  solution  of  phosphate  of  potash        ...  5  parts 

Saturated  solution  of  sulphate  of  soda     ....  5  parts 

Water loo  parts 

This  formula  is  suitable  for  general  maceration.  (Arch.f. 
Mikr.  Anat.,  1885.) 

Bern's  method  of  reconstructing  objects  from  microscopic  sec- 
tions. —  Dr.  G.  Born l  describes  in  detail  a  very  ingenious  method 

1  Arch.  f.  Mikr.  Anat.,  XXII,  p.  584,  1883  ;  Jour.  Roy.  Mic.  Soc.,  p.  634,  1884; 
Amer.  Nat..  XVIII,  p.  446,  1884. 


238  BIOLOGICAL  LABORATORY   METHODS 

of  constructing  models  of  objects  from  serial  sections.  By  aid 
of  the  camera  lucida  the  outlines  of  the  sections  are  transferred  to 
wax  plates,  which  are  then  cut  out  so  as  to  correspond  in  out- 
lines as  well  as  dimensions  to  the  sections  equally  magnified  in 
all  three  directions.  With  plates  thus  prepared,  it  is  only 
necessary  to  put  them  together  in  the  proper  order  to  obtain  a 
complete  model.  The  method  is  simple  and  extremely  useful, 
especially  in  investigating  objects  with  complex  internal  cavities. 
Born  has  made  use  of  the  method  in  studying  different  parts 
of  the  vertebrate  head ;  Swirski,  in  elucidating  the  develop- 
ment of  the  shoulder  girdle  of  the  pike ;  Stohr,  in  tracing  the 
development  of  the  skull  of  the  Amphibia  and  Teleostei ;  and 
Uskow,  in  studying  the  development  of  the  body  cavity,  the  dia- 
phragm, etc. 

Born  makes  use  of  three  rectangular  tin  boxes  of  equal  sizes, 
each  measuring  270  x  230  x  2\  mm.  The  sections  should  be 
made  about  -^  mm.  thick  (never  thinner  than  -fa  mm.).  If  we 
desire  to  construct  a  model  of  an  object  from  serial  sections 
-g1^  mm.  thick  which  shall  be  magnified  60  diameters,  then  the 
wax  plates  must  be  made  sixty  times  as  thick  as  the  sections, 
i.e.  2  mm.  thick. 

The  surface  of  a  plate  that  could  be  made  in  a  box  of  the 
above  dimensions  contains  62,100  sq.  mm.;  and  the  volume  of 
such  a  plate  2  mm.  thick  would  therefore  be  124.2  cc.  The 
specific  gravity  of  common  raw  beeswax  amounts  to  0.96  to 
0.97.  For  use,  it  requires  only  to  be  melted  and  a  little  turpen- 
tine added  to  make  it  more  flexible.  Thus  prepared,  its  specific 
gravity  is  about  0.95  ;  and  this  number  has  been  sufficiently 
accurate  in  all  cases.  The  weight  of  the  wax  required  to  make 
one  plate  of  the  above  size  will  accordingly  be  117.99  grammes. 
The  wax  having  been  weighed  and  melted,  the  tin  box  is  first 
filled  i-J-  cm.  deep  with  boiling  water,  and  then  the  melted  wax 
poured  upon  the  water.  If  the  water  and  wax  are  quite  hot,  the 
wax  will  generally  spread  evenly  over  the  surface ;  if  gaps 
remain,  they  can  be  filled  out  by  the  aid  of  a  glass  slide  drawn 
over  the  wax.  As  soon  as  the  plate  has  stiffened,  and  while  it 


MACERATION  239 

is  still  soft,  it  is  well  to  cut  it  free  from  the  walls  of  the  tin  box, 
as  further  cooling  of  the  water  and  the  box  might  cause  it  to 
split.  By  the  time  the  water  becomes  tepid,  the  plate  can  be 
removed  from  the  water  to  some  flat  support,  and  left  until 
completely  stiffened. 

The  outlines  of  the  sections  are  transferred  to  the  plate  in  the 
following  manner :  A  piece  of  blue  paper  is  placed  on  the  plate 
with  the  blue  side  turned  toward  the  wax,  and  above  this  is 
placed  a  sheet  of  ordinary  drawing-paper.  The  outlines  are 
drawn  on  the  latter  by  the  aid  of  the  camera  lucida,  and  at  the 
same  time  blue  outlines  are  traced  on  the  wax  plate.  The  plate 
can  then  be  laid  on  soft  wood  and  cut  out  by  the  assistance  of  a 
small  knife.  Thus  a  drawing  and  a  model  of  each  section  are 
prepared.  The  plates  thus  prepared  can  be  put  together  in  the 
proper  order,  and  fastened  by  the  aid  of  a  hot  spatula  applied 
to  the  edges. 

Professor  H.  Strasser l  gives  an  improvement  and  a  simplifi- 
cation of  the  Born  plate  model  system,  consisting  in  the  adop- 
tion of  transparent  plates,  which  are  also  thinner  than  any 
hitherto  used. 

The  apparatus  required  in  the  preparation  of  the  plates  are 
an  iron  roller,  4  cm.  in  diameter  and  30  cm.  in  length  ;  a  water- 
bath  for  keeping  the  wax  at  a  temperature  of  60°  C. ;  some 
strips  of  tin  and  brass  from  0.2  to  5.0  mm.  thick,  and  a  large 
smooth  lithographic  stone. 

In  preparing  the  wax  plates,  a  piece  of  the  still  warm  wax  is 
kneaded  out  in  the  hands  as  flat  as  possible,  and  having  been 
placed  between  two  leaves  of  parchment  paper  kept  moistened 
by  turpentine,  is  rolled  out  by  means  of  the  roller  previously 
warmed.  The  thickness  of  the  lamella  is  regulated  by  the 
choice  of  the  metal  strips  placed  along  the  sides  of  the  paper. 

When  a  perfectly  flat  layer  has  been  thus  rolled  out,  the 
parchment  paper  is  stripped  off  and  the  paper  dried  between 
filter-papers.  To  the  surface  of  these,  wax  plates  are  made  to 
adhere  by  means  of  gum,  for  which  purpose  flour  is  first  rubbed 

1  Zeitschr.f.  wiss.  Mikr.,  Vol.  Ill,  p.  179, 1886 ;  Jour.  Roy.  Mic.  Soc.t  p.  171,  1887. 


240  BIOLOGICAL   LABORATORY   METHODS 

into  the  plate,  or  by  melting  it  in  by  means  of  a  hot  roller.  The 
plates  thus  produced  are  of  fair  size,  and  from  1  to  J  mm.  thick. 
In  preparing  wax-paper  plates  to  which  the  section  sketch  is 
attached,  a  very  similar  procedure  is  carried  out.  One  of  the 
leaves  of  the  tracing-paper  is  placed  on  the  lithographic  stone 
damped  with  turpentine.  On  the  other  side  is  laid  a  strip  of 
metal,  then  the  wax  is  spread  over  the  surface,  and  the  sec- 
ond leaf  of  tracing-paper  having  been  adjusted,  a  flat  lamina  is 
produced  by  rolling,  as  before,  with  the  heated  roller.  The  thick- 
ness of  these  plates,  paper  and  all,  may  not  exceed  0.2  mm. 
Although  firm  in  consistency,  they  are  perfectly  flexible,  and 
are  cut  with  sharp  knives  or  scissors  quite  easily. 

Methods  for  the  Preservation  of  Marine  Organisms J 

Professor  Playfair  McMurrich,  in  writing  about  the  work  at 
the  Naples  Zoological  Station,  gives  the  following  account  of 
the  experiments  concerning  the  preservation  of  delicate  marine 
organisms  made  by  the  conservator  of  the  station,  Salvator  Lo 
Bianco  :  "  The  last  number  of  the  Naples  Mittheilungen*  con- 
tains a  full  description  of  the  methods  found  most  successful 
for  the  preservation  of  the  various  forms  which  occur  at  Naples, 
and  which  are  undoubtedly  applicable  to  the  similar  forms  found 
upon  our  own  coasts.  An  abstract  is  given  in  the  following 
pages.  It  must  be  fully  understood,  however,  that  much  de- 
pends upon  the  skill  of  the  preparator,  and  that  want  of  care 
and  patience  will  frequently  counteract  all  the  advantages  to  be 
derived  from  a  good  method.  All  who  have  had  the  opportu- 
nity of  examining  specimens  prepared  by  Lo  Bianco  can  appre- 
ciate readily  the  great  advantages  which  may  result  from  a 
careful  application  of  his  methods,  and  can  perceive  how 
greatly  we  are  indebted  to  him  and  to  Professor  Dohrn  for 
their  publication. 

"  Alcohol  is,  of  course,  indispensable  as  a  preservative  fluid, 
but  certain  precautions  are  necessary  in  its  use.  Except  in  a 

1  Amer.  Nat.,  XXIV,  p.  856,  1890. 

2  MUtheil.  Zool.  Stat.,  Neapel,  IX,  p.  435,  1890. 


MACERATION  24! 

few  cases  it  is  unnecessary  to  use  it  in  its  full  strength,  70 
per  cent  being  quite  sufficient  for  preservation,  and  producing 
much  less  contraction  and  fragility  in  delicate  organisms. 
Strong  alcohol  should  be  reduced  with  distilled  water  to  the 
desired  strength,  ordinary  spring-water  frequently  containing 
a  sufficient  amount  of  carbonate  of  lime  and  other  substances 
in  solution  to  give  a  cloudy  precipitate,  after  a  time,  which 
may  effectually  destroy  the  appearance  of  the  specimen. 

"  Furthermore,  delicate  organisms  should  first  be  placed  in 
weak  alcohol  (35  to  50  per  cent)  for  from  two  to  six  hours,  the 
changing  of  the  fluids  being  effected  by  a  siphon,  a  small  quan- 
tity of  the  weak  alcohol  being  withdrawn  and  stronger  added, 
until  finally  the  desired  strength  is  obtained.  With  delicate 
gelatinous  structures  the  increase  in  the  strength  of  the  alcohol 
should  be  as  gradual  as  possible.  In  many  cases  it  is  necessary 
to  use  a  hardening  or  fixing  agent  before  the  final  consignment  to 
alcohol,  which  is  principally  useful  as  a  preservative.  The  most 
useful  fixing  agents,  according  to  Lo  Bianco,  are  as  follows  :  — 

"  Chromic  acid.  —  One  per  cent  in  fresh  water.  Objects 
should  not  remain  in  the  fluid  longer  than  is  necessary  to  fix 
them,  as  they  are  apt  to  become  brittle.  Subsequently  they 
should  be  well  washed  with  distilled  water  to  prevent  the  forma- 
tion of  precipitates  when  placed  in  alcohol,  and  also  to  prevent 
their  taking  on  too  green  a  tinge  from  the  reduction  of  the  acid. 

"Acetic  acid,  concentrated,  kills  rapidly  contractile  animals,  but 
must  be  used  with  caution,  as  it  produces  a  softening  of  the 
tissues  if  they  are  subjected  for  too  long  a  time  to  its  action. 

"  Osmic  acid.  —  One  per  cent  solution  hardens  gelatinous  forms 
well  and  preserves  their  transparency,  but  its  prolonged  action 
renders  the  objects  brittle  and  gives  a  dark-brown  tint.  Objects 
hardened  in  it  should  be  well  washed  in  distilled  water  before 
being  placed  in  alcohol. 

"  Lactic  acid.  —  One  part  to  1000  parts  of  sea-water  fixes  larvae 
and  gelatinous  forms  well. 

"  Corrosive  sublimate.  —  Saturated  solution  in  fresh  or  sea 
water ;  may  be  used  either  hot  or  cold.  It  acts  quickly,  and 
R 


242  BIOLOGICAL   LABORATORY   METHODS 

preserves  admirably  for  histological  purposes.  It  is  especially 
good  combined  with  copper  sulphate,  acetic  acid,  or  chromic 
acid.  Objects  hardened  in  it  should  be  subsequently  washed  in 
distilled  water  and  in  iodized  alcohol  (the  recipe  for  which  is  given 
below),  to  remove  all  traces  of  the  sublimate,  which  in  alcohol 
crystallizes  out  in  the  tissues  of  the  organisms  and  so  injures 
the  preparation. 

"  Bichromate  of  potassium.  —  Five  per  cent  solution  in  distilled 
water  hardens  gelatinous  organisms  slowly,  without  rendering 
them  fragile.  It  gives,  however,  a  precipitate  in  alcohol,  and 
discolors  the  specimen.  The  discoloration,  however,  may  be 
removed  by  adding  to  the  alcohol  a  few  drops  of  concentrated 
sulphuric  acid. 

"  Copper  sulphate.  —  Five  per  cent,  or  10  per  cent,  solution  in 
distilled  water,  used  either  alone  or  in  combination  with  cor- 
rosive sublimate,  kills  larvae  and  delicate  animals  without  dis- 
tortion. The  objects  should  be  subsequently  repeatedly  washed 
with  water  to  remove  all  traces  of  the  salt,  otherwise  crystals 
will  form  when  the  object  is  placed  in  alcohol. 

"  Various  combinations  of  these  reagents  are  especially  useful, 
and  some  of  those  most  serviceable  are  given  below :  — 

Alcohol  and  chromic  acid. 

70  per  cent  alcohol  ^ 

.         .        .     >  .....  equal  parts 

I  per  cent  chromic  acid  J 

Alcohol  and  hydrochloric  acid. 

50  per  cent  alcohol .         .     100  cc. 

Hydrochloric  acid,  concentrated  .         .         .         .         .         5  cc. 

Iodized  alcohol. 

35  per  cent  or  70  per  cent  alcohol 100  cc. 

Tincture  of  iodine 2.5  cc. 

Chrom-acetic  acid,  No.  I. 

i  per  cent  chromic  acid       .         .         ...         .         .     100  cc. 

Concentrated  acetic  acid      .......         5  cc< 

Chrom-acetic  acid,  No.  2. 

Concentrated  acetic  acid 100  cc. 

I  per  cent  chromic  acid 10  cc. 


MACERATION  243 

Chrom-osmic  acid. 

i  per  cent  chromic  acid       .......     100  cc. 

i  per  cent  osmic  acid  ........         2  cc. 

Chrom-picric  acid. 

i  per  cent  chromic  acid  ") 

TT i  •       i       ,     •  i  i-     •       -j  r     •        •        •        •          equal  parts 

Klemenberg's  picro-sulphunc  acid  ) 

Copper  sulphate  and  corrosive  sublimate. 

10  per  cent  solution  copper  sulphate 100  cc. 

Saturated  solution  corrosive  sublimate          .         .         .  10  cc. 

Potassium  bichromate  and  osmic  acid. 

5  per  cent  solution  potassium  bichromate    ....     100  cc. 
I  per  cent  osmic  acid  .         .         .         ...         .         .         2  cc. 

Corrosive  sublimate  and  acetic  acid. 

Saturated  solution  of  corrosive  sublimate     ....     loo  cc. 
Concentrated  acetic  acid 50  cc. 

Corrosive  sublimate  and  chromic  acid. 

Saturated  solution  of  corrosive  sublimate  /  .        '.  .     100  cc. 

I  per  cent  chromic  acid        .......       50  cc. 

"  Frequently  great  difficulty  is  experienced  in  killing  an  animal 
without  producing  a  considerable  amount  of  contraction,  and  in 
the  case  of  elongated  forms,  such  as  Nemerteans,  and  other 
worms,  without  causing  them  to  coil  up  or  become  twisted.  To 
avoid  this  it  is  expedient  to  narcotize  the  animals  before  killing 
them,  and  for  this  purpose  Lo  Bianco  recommends  immersion 
in  weak  alcohol.  He  uses  generally  a  mixture  of  sea-water 
100  cc.  and  absolute  alcohol  5  cc.  In  other  cases  70  per  cent 
alcohol  may  be  carefully  poured  upon  water  in  which  the  speci- 
men lies,  so  that  it  forms  a  layer  at  the  surface.  It  will  gradually 
mix  with  the  subjacent  water,  and  in  the  course  of  a  few  hours 
will  narcotize  the  animal,  so  that  it  may  be  treated  with  fixing 
reagents  without  fear  of  contraction. 

"Chloral  hydrate,  i  to  2  parts  sea-water,  is  also  efficient  as  a 
narcotizing  agent,  and  has  the  advantage  of  allowing  a  recovery 
of  the  animal,  if  there  should  be  necessity  for  it,  by  placing  it  in 
fresh  sea-water.  For  some  sea-anemones  tobacco  smoke  is  use- 
ful, the  smoke  being  conducted  by  a  V-shaped  tube  into  a  bell- 


244  BIOLOGICAL  LABORATORY   METHODS 

jar  covering  the  vessel  of  sea-water  in  which  is  the  anemone. 
Certain  of  these  reagents  will  prove  most  satisfactory  with  some 
animals,  others  with  others.  Lo  Bianco  details  the  best  method 
for  treating  the  various  forms  in  a  second  portion  of  his  paper, 
and  an  account  of  some  of  his  methods  of  procedure,  so  far  as 
they  concern  forms  which  resemble  those  found  upon  our  coast, 
may  now  be  presented. 

"Sponges.  —  Direct  immersion  in  70  per  cent  alcohol,  with  sub- 
sequent renewal  of  the  fluid,  is  recommended  for  the  majority 
of  forms.  To  avoid  contraction  in  the  case  of  the  Halisarcidae, 
they  should  be  left  for  half  an  hour  in  i  per  cent  chromic  acid, 
or  in  concentrated  solution  of  corrosive  sublimate  for  fifteen 
minutes.  To  prepare  dried  specimens  the  sponges  should  be 
washed  in  fresh  water  for  a  few  hours,  and  then  allowed  to 
remain  in  ordinary  alcohol  for  a  day,  after  which  they  may  be 
dried  in  the  sun. 

"Anthozoa.  —  The  first  care  must  be  to  place  the  forms  belong- 
ing to  this  group  in  fresh  salt  water,  to  allow  them  to  expand, 
a  result  which  may  not  be  obtained  until  the  following  day  in 
some  cases.  Alcyonarians  should  be  killed  with  chrom-acetic 
solution  No.  2,  withdrawing  the  water  in  which  they  lie,  until 
there  is  left  just  enough  to  cover  them,  and  then  adding  a  volume 
of  the  chrom-acetic  solution  double  that  of  the  sea-water.  The 
animals  should  be  removed  from  this  mixture  the  moment  they 
are  killed,  since  the  acid  will  quickly  attack  the  calcareous  spic- 
ules,  which  are  important  for  the  identification  of  the  Alcyonaria, 
and  placed  in  35  per  cent  or  50  per  cent  alcohol,  it  being  well  to 
inject  the  alcohol  into  the  mouth  of  the  polyps  to  keep  them 
freely  expanded.  The  preparation  should  finally  be  preserved 
in  70  per  cent  alcohol. 

"Regarding  the  Actinians  no  definite  rule  for  preservation  can 
be  given.  Much  of  the  success  of  the  preparation  depends  on 
the  form  employed,  some  species  contracting  much  less  readily 
and  less  perfectly  than  others.  Some  may  be  killed  in  a  fair 
condition  by  pouring  over  them  boiling  corrosive  sublimate,  and 
then,  before  consigning  them  to  alcohol,  treating  for  a  few 


MACERATION  245 

minutes  with  one-half  per  cent  chromic  acid.  This  method  may 
be  employed  with  small  forms  such  as  Aiptasia.  Narcotization 
may  be  tried  with  others.  For  this  purpose,  remove  from  the 
vessel  in  which  the  animals  are  contained  two-thirds  of  sea- 
water,  and  replace  it  with  a  2  per  cent  solution  of  chloral  hydrate. 
After  a  few  minutes  the  fluid  is  again  removed,  and  cold  con- 
centrated corrosive  sublimate  solution  is  poured  in.  Tobacco 
smoke  in  some  cases,  as  with  Adamsia,  will  act  satisfactorily,  if 
followed  with  vapor  of  chloroform  for  two  or  three  hours,  after 
which  the  animals  may  be  killed  in  chrom-acetic  solution  No.  2, 
and  hardened  in  one-half  per  cent  chromic  acid. 

"Edwardsia  may  be  narcotized  by  gradually  adding  70  per  cent 
alcohol  to  sea-water  in  which  they  are,  and  subsequently  may  be 
killed  with  hot  corrosive  sublimate. 

"Cerianthus  should  be  killed  with  concentrated  acetic  acid, 
placing  it  as  soon  as  possible  in  weak  alcohol,  in  which  it  should 
be  suspended,  so  that  the  tentacles  may  float  freely  —  if  neces- 
sary, disentangle  them. 

"Corals  should  be  allowed  to  expand  fully,  and  then  should  be 
killed  with  boiling  solution  of  corrosive  sublimate  and  acetic 
acid  used  in  volume  equal  to  that  of  sea-water  containing  the 
coral.  The  colony  should  then  be  transferred  to  35  per  cent 
alcohol,  some  of  this  fluid  being  injected  into  the  mouth  of  each 
polyp.  The  injection  should  be  repeated  at  every  change  of 
the  alcohol,  and  the  specimens  should  be  preserved  in  70  per 
cent  alcohol  after  washing  them  well  in  iodized  alcohol. 

"Hydromedusae.  —  For  the  hydroid  colonies  the  best  fixing 
reagent  is  hot  corrosive  sublimate.  The  smaller  Tubularian 
medusae  should  be  killed  either  in  the  mixture  of  corrosive  sub- 
limate and  acetic  acid  or  in  Kleinenberg's  picro-sulphuric  acid. 
Larger  forms  may  be  fixed  with  concentrated  acetic  acid,  and 
then  allowed  to  fall  into  a  tube  containing  the  alcohol  and  chro- 
mic acid  mixture,  in  which  they  are  gently  agitated  and  allowed 
to  remain  for  fifteen  minutes,  after  which  they  should  be  trans- 
ferred to  35  per  cent  alcohol,  and  gradually  carried  to  70  per 
cent  alcohol. 


246  BIOLOGICAL  LABORATORY   METHODS 

"Small  Campanularian  medusae,  e.g.  Eucope  and  Obelia,  may 
be  killed  in  the  mixture  of  copper  sulphate  and  corrosive  subli- 
mate. ^Equorea  should  be  killed  with  concentrated  acetic  acid, 
and  immediately  transferred  to  chrom-osmic  mixture  for  fifteen 
to  thirty  minutes.  The  same  method  answers  for  Cunina,  while 
Liriope  should  be  treated  at  once  with  chrom-osmic  from  five  to 
twenty  minutes. 

"Scyphomedusae  are  the  best  fixed  with  i  per  cent  osmic  acid, 
to  the  action  of  which  they  are  subjected  until  they  assume  a 
pale  brown  tint.  They  should  then  be  thoroughly  washed  with 
fresh  water  before  being  placed  in  35  per  cent  alcohol,  and 
should  be  finally  preserved  in  70  per  cent. 

"Siphonophores.  —  The  forms  of  this  group  should  be  preserved 
soon  after  capture,  and  specimens  in  good  condition  should  be 
selected.  Agalma  and  similar  forms  should  be  killed  in  a 
mixture  of  copper  sulphate  and  sublimate,  which  should  be  used 
in  volume  equal  to  or  double  that  of  the  sea-water  in  which  the 
animal  floats.  The  mixture  should  be  poured  in  rapidly,  and 
not ^ over  the  animal.  When  killed  the  specimen  should  be 
carefully  lifted  upon  a  large  horn  spatula,  and  transferred  to 
35  per  cent  alcohol  for  a  few  hours,  and  then  placed  in  70  per 
cent  alcohol.  It  is  recommended  to  preserve  the  animals  in 
tubes  just  large  enough  to  contain  the  specimens,  and  placed  in 
a  second  larger  tube.  In  this  way  evaporation  of  the  alcohol  is 
prevented,  and  also  injury  to  the  specimen  from  movements  of 
the  liquid  is  avoided. 

"Physalia  should  be  placed  in  a  cylinder  filled  with  sea-water, 
the  animal  being  lifted  by  the  pneumatophore.  When  well  ex- 
panded, it  is  killed  by  pouring  over  it  the  sublimate  and  acetic 
acid  mixture  (one-quarter  the  volume  of  the  sea-water),  and  when 
dead  is  transferred  to  a  cylinder  containing  one-half  per  cent 
chromic  acid,  and  then,  after  twenty  minutes,  to  50  per  cent 
alcohol,  and  finally  to  70  per  cent  alcohol. 

"Velella  may  be  killed  with  chrom-picric  or  sublimate  and 
chromic  acid  mixture,  and  after  a  few  minutes  should  be  trans- 
ferred to  weak  alcohol.  Porpita  may  be  fixed  by  dropping 


MACERATION  247 

Kleinenberg's  picro-sulphuric  acid  into  the  vessel  in  which  it  is 
contained,  and  when  the  blue  color  begins  to  change  to  red  it 
should  be  transferred  to  Kleinenberg's  fluid,  and  after  fifteen 
minutes  to  weak  alcohol. 

"Diphyes  may  be  killed  expanded  by  hot  corrosive  sublimate. 

"Ctenophora  may  be  killed  by  throwing  them  into  the  chrom- 
osmic  mixture,  where  they  should  remain  for  fifteen  to  sixteen 
minutes,  according  to  the  size,  and  then  gradually  passing  them 
through  alcohol  to  70  per  cent.  A  mixture  composed  of  pyro- 
ligneous  acid,  concentrated,  i  vol.  ;  corrosive  sublimate  solution, 
2  vols. ;  one-half  per  cent  chromic  acid,  i  vol.,  is  also  recom- 
mended as  a  fixative. 

"Echinodermata.  —  Starfish  may  be  prepared  with  the  ambu- 
lacral  feet  in  full  distension  by  allowing  them  to  die  in  20  to  30 
per  cent  alcohol.  Echinoids  may  be  placed  in  a  small  quantity 
of  water,  and  killed  with  chrom-acetic  mixture  No.  2,  being 
removed  from  it  as  quickly  as  possible,  as  acid  corrodes  the 
test.  To  preserve  the  internal  parts  it  is  necessary  to  make  two 
opposite  openings  in  the  test,  so  that  the  alcohol  may  penetrate 
the  interior  readily. 

"Holoth urians  —  such  as  Thyone  and  Cucumaria  —  after  the 
tentacles  are  fully  expanded,  should  be  seized  a  little  below  the 
bases  of  the  tentacles  by  forceps,  using  a  slight  pressure,  and 
the  anterior  portion  of  the  body  should  then  be  immersed  in 
concentrated  acetic  acid.  Alcohol  (90  per  cent)  should  then  be 
injected  into  the  mouth,  and  the  specimens  placed  in  70  per 
cent  alcohol.  The  injection  should  be  repeated  each  time  the 
alcohol  is  changed. 

"Synapta  should  be  fixed  by  immersion  in  a  tube  containing  a 
mixture  of  equal  parts  of  sea-water  and  ether  (or  chloroform), 
where  they  remain  completely  expanded.  They  should  then 
be  washed  for  a  short  time  in  fresh  water,  and  passed  into 
alcohol,  taking  care  to  increase  the  strength  of  this  very  grad- 
ually. 

"Vermes.  —  Cestodes,  Trematodes,  Turbellaria,  as  well  as 
Nemathelminths,  are  most  satisfactorily  killed  with  corrosive 


248  BIOLOGICAL   LABORATORY   METHODS 

sublimate,  either  cold  or  hot.  Sagitta,  however,  succeeds  best 
in  copper  sulphate  and  sublimate  or  chrom-osmic  mixture. 

"Nemerteans  should  be  narcotized  in  a  solution  of  chloral 
hydrate  in  sea-water  i  per  cent,  where  they  should  remain  for 
six  to  twelve  hours.  They  are  then  to  be  hardened  in  alcohol. 

"Gephyreans  may  be  narcotized  with  i  per  cent  solution  of 
chloral  hydrate  in  sea-water,  or  in  alcoholized  sea-water,  three 
to  six  hours ;  or  may  be  killed  at  once  in  one-half  per  cent 
chromic  acid,  which  last  method  may  be  applied  to  Hirudinea. 

"Chcetopods  are  best  narcotized  in  sea-water  containing  5  per 
cent  of  absolute  alcohol,  or  by  adding  gradually  to  the  surface 
of  the  sea-water  in  which  they  are  contained,  a  mixture  of  glyce- 
rin i  part,  70  per  cent  alcohol  2  parts,  and  sea-water  2  parts, 
hardening  them  subsequently  in  alcohol.  Chaetopterus  is  best 
killed  with  i  per  cent  chromic  acid,  in  which  they  should  remain 
for  half  an  hour ;  while  the  Hermellidae,  ^Ephroditidae,  and  the 
Eunicinae  may  be  killed  in  cold  corrosive  sublimate.  Some  of 
these,  such  as  Diopatra,  may,  however,  be  narcotized  in  alcohol- 
ized sea-water. 

"Serpulidce,  before  treatment  with  corrosive  sublimate,  should 
be  narcotized  in  i  per  cent  chloral  hydrate,  which  causes  them 
to  protrude  wholly  or  partly  from  their  tubes. 

"Crustacea.  —  Cladocera,  Copepods,  and  Schizopods  may  be 
killed  in  corrosive  sublimate  dissolved  in  sea-water.  Ostrapods 
may  be  thrown  at  once  into  70  per  cent  alcohol.  Cirripedes  die 
expanded  in  35  per  cent  alcohol ;  and  if  some  specimens  contract, 
it  is  easy  to  draw  out  the  cirri  with  forceps.  Amphipods  and 
Isopods  may  pass  directly  into  70  per  cent  alcohol,  except  the 
Bopyrids  and  Entoniscids,  which  should  be  killed  in  a  mixture 
of  equal  parts  of  90  per  cent  alcohol  and  sublimate  solution. 

"To  avoid  casting-off  of  the  appendages  of  the  Decapods,  they 
should  be  allowed  to  die  in  fresh  water,  care  being  taken  not  to 
allow  them  to  remain  in  it  longer  than  is  necessary,  as  it  causes 
a  distortion  of  the  membranous  appendages. 

"Pycnogonids  will  die  in  one-half  per  cent  chromic  acid  with 
the  appendages  fully  extended. 


MACERATION  249 

"Mollusca.  —  Lamellibranchs,  Prosobranchs,  and  Heteropods 
should  be  narcotized  in  alcoholized  sea-water.  To  avoid  the 
closure  of  the  valves  of  Lamellibranchs  on  immersion  in  70  per 
cent  alcohol,  little  plugs  of  wood  should  be  placed  between  the 
margins  of  the  valves.  The  same  result  may  be  effected  in 
the  case  of  Prosobranchs  by  tying  the  internal  edge  of  the  oper- 
culum  to  the  shell. 

"Of  the  Opisthobranchs  the  aeolidae  may  be  best  preserved 
by  pouring  over  them  concentrated  acetic  acid  in  volumes  equal 
to  or  double  that  of  the  sea-water  containing  them.  Dorids 
should  first  be  narcotized  by  gradually  adding  70  per  cent 
alcohol  to  their  sea-water,  and  then  killed  with  concentrated 
acetic  acid  or  boiling  sublimate.  The  larger  forms  may  be 
killed  in  i  to  5  per  cent  chromic  acid. 

"Pteropods  are  preserved  well  in  Perenyi's  fluid  for  fifteen 
minutes,  whence  they  are  passed  to  50  per  cent  alcohol. 
Gymnosomatous  forms  should  be  first  narcotized  in  i  per  cent 
chloral  hydrate,  and  then  killed  in  acetic  acid  or  sublimate. 

"Decapod  Cephalopods  may  be  fixed  directly  in  70  per  cent 
alcohol,  making  an  opening  on  the  ventral  surface  to  allow  the 
alcohol  to  reach  the  internal  parts*. 

"Bryozoa.  —  The  genera  Pedicellina  and  Loxosoma  may  be  left 
for  one  hour  in  a  i  per  cent  chloral  hydrate,  and  then  killed  • 
with  cold  corrosive  sublimate,  washing  them  immediately  after- 
ward. Some  species  of  Bugula  give  good  results  when  the 
expanded  animals  are  suddenly  killed  by  pouring  over  them  hot 
corrosive  sublimate.  With  other  forms  it  is  possible  to  pre- 
serve them  well  expanded  by  adding  70  per  cent  alcohol  grad- 
ually to  the  surface  of  the  water  in  which  they  are,  or  by 
narcotizing  first  in  weak  chloral  hydrate  or  in  alcoholized  sea- 
water.  The  results,  however,  are  uncertain,  and  depend  largely 
on  the  skill  of  the  preparator.  Brachiopoda  may  be  treated  in 
the  same  manner  as  Lamellibranchs. 

"Tunicates.  —  Clavellina,  Perophora,  and  Molgula  may  be  killed 
with  the  orifices  expanded  by  immersing  them  in  i  per  cent 
chloral  hydrate  for  6  to  12  hours,  They  should  then  be  killed 


25O  BIOLOGICAL  LABORATORY  METHODS 

in  chromic  acetic  mixture  No.  2,  and  quickly  transferred  to 
i  per  cent  chromic  acid,  injecting  some  of  the  fluid  into  the 
body.  After  half  an  hour  they  should  be  transferred  to  35  per 
cent  alcohol,  the  injection  repeated,  and  finally  to  70  per  cent 
alcohol.  Other  simple  forms  may  be  treated  in  the  same  man- 
ner, or  may  require  the  2  per  cent  solution  of  chloral  hydrate, 
or  may  be  killed  by  pouring  a  little  i  per  cent  chromic  acid  on 
the  surface  of  the  water  in  which  they  are,  subsequently  hard- 
ening in  i  per  cent  chromic  acid.  The  method  recommended 
for  Perophora  may  be  employed  for  compound  Ascidians,  using, 
however,  corrosive  sublimate  instead  of  the  chrom-acetic  acid 
mixture. 

"Salpae  vary  considerably  in  consistency,  according  to  the 
species,  and  different  methods  are  consequently  required.  The 
denser  forms,  such  as  S.  zonaria,  should  be  placed  in  a  mixture 
of  100  cc.  fresh  water  and  10  cc.  concentrated  acetic  acid, 
where  they  should  remain  for  fifteen  minutes.  They  should 
then  be  washed  in  fresh  water  for  ten  minutes,  and  pass  gradu- 
ally into  fresh  alcohol ;  while  the  soft  forms,  such  as  S.  pinnata 
and  maxima,  should  be  placed  in  chrom-osmic  mixture  for 
fifteen  to  sixty  minutes,  then  washed  in  fresh  water  and  trans- 
ferred to  weak  alcohol. 

"Fishes.  —  Amphioxus  will  die  with  the  buccal  cirri  distended 
in  sea-water  alcoholized  to  10  per  cent.  They  should  then  be 
transferred  to  50  per  cent  alcohol,  and  gradually  to  70  per 
cent. 

"Other  forms  may  be  preserved  in  alcohol  (70  per  cent),  taking 
care  to  make  a  ventral  incision,  and  also  to  inject  the  alcohol 
and  to  renew  it  frequently  at  first.  If  it  is  wished  to  preserve 
some  of  the  larger  Selachians  for  some  months,  in  order  to 
prepare  at  leisure  the  skeleton,  the  intestines  should  be  removed, 
and  the  animals  placed  in  10  per  cent  solution  of  salt. 

"Elasmobranch  embryos  may  be  fixed  in  corrosive  sublimate, 
leaving  them  in  the  solution  for  five  to  fifteen  minutes,  after- 
ward washing  well  in  iodized  alcohol.  Embryos  of  Torpedo 
with  the  yolk  were  preserved  by  immersing  in  a  mixture  of 


MACERATION  251 

equal  parts  of  i  per  cent  of  chromic  acid  and  corrosive  subli- 
mate for  fifteen  minutes,  and  then  transferring  to  alcohol. 
Transparent  fish  eggs  may  be  preserved  for  the  purpose  of 
demonstration  by  subjecting  them  for  a  few  minutes  to  the 
action  of  the  alcohol  and  hydrochloric  acid  mixture,  and  then 
transferring  them  to  pure  alcohol." 

There  is  another  method  which  is  sometimes  used  in  this 
country  for  the  microscopic  study  of  marine  forms,  which  is 
based  upon  the  gradual  displacement  of  the  sea-water  in  which 
the  animal  is  living  with  formaldehyde,  and  the  latter  with 
gelatin,  in  which  the  animal  is  finally  mounted.  This  work 
may  be  accomplished  by  using  a  glass  slip,  on  which  the  animal 
is  placed  in  a  drop  of  sea-water.  Over  the  water  is  adjusted  a 
cover-glass  resting  on  wax  feet,  and  the  formaldehyde  is  drawn 
under  while  the  sea-water  is  gradually  extracted  from  the  other 
side  of  the  cover.  The  formaldehyde  is  extracted  with  gelatin 
in  same  manner,  and  then  the  cover-glass  is  sealed  permanently. 


CHAPTER   XVI 

POLARIZATION    OF   LIGHT   AND    ITS   APPLICATION    TO 
BIOLOGICAL    INVESTIGATIONS 

What  is  polarized  light  ?  To  answer  this  question  fully  will 
require  a  lengthy  discussion  of  physical  laws  and  phenomena 
bearing  on  the  subject,  and  this  will  not  be  desirable  for  a  book 
of  this  character  ;  but  it  will  be  possible  to  enlighten  the  student 
by  giving  some  of  the  general  principles  on  the  topic  of  polar- 
ized light. 

The  law  governing  the  transmission  of  light  states  that  the 
ether,  or  the  substance  which  permeates  all  space,  is  vibrated 
under  the  influence  of  the  light-emitting  body,  and  that  these 
light  waves,  or  vibrations,  take  place  at  right  angles  to  the 
direction  toward  which  the  light  is  travelling.  These  vibra- 
tions, moreover,  are  all  along  the  radii  of  the  beam  of  light. 

To  polarize  the  beam  of  light,  therefore,  it  is  necessary  to 
reduce  the  vibrations  to  two  planes  by  overcoming  the  vibra- 
tions in  all  other  directions.  The  beam  that  comes  directly 
from  the  sun,  and  which  is  not  polarized,  can  be  bent  in  any 
direction  by  the  interposition  of  a  reflecting  surface  ;  but  if  this 
beam  is  first  passed  through  a  medium  which  polarizes  the  light, 
the  reflecting  surface  can  then  bend  the  rays  only  in  two  direc- 
tions, because  the  vibrations  have  been  overcome  in  all  other 
diameters  of  the  original  beam  except  in  two.  These  two  polar- 
ized vibratory  conditions  of  the  ether  are  termed  the  "  ordi- 
nary "  and  the  "  extraordinary  "  rays. 

If  a  piece  of  Iceland  spar,  or  carbonate  of  lime,  is  placed  on 
a  sheet  of  paper  containing  writing,  two  images  of  each  letter 
are  seen,  which  shows  that  the  spar  has  divided  the  beam  of 
light  coming  from  the  writing  into  two  planes  of  vibrations, 

252 


POLARIZATION  OF  LIGHT  253 

each  having  the  power  of  transmitting  its  image  of  the  letter 
in  different  planes.  An  analysis  of  this  action  of  the  crystal 
will  show  that  these  rays  are  polarized  at  right  angles  to  each 
other. 

In  order  to  use  this  property  of  the  calcium  carbonate  in 
connection  with  the  microscope,  we  must  dispense  with  one  of 
these  rays,  and  this  is  accomplished  by  cutting  the  crystal  in 
half  (Fig.  120),  along  the  line  BD,  and  then  cementing  the  halves 
together  again  by  means  of  Canada  balsam.  When  the  beam  of 
light  is  passed  through  the  crystal  thus  prepared,  the  ordinary  ray 


FlG.  120. —  Iceland  Spar,  showing  Polarized  Light. 

strikes  the  Canada  balsam  at  such  an  oblique  angle l  that  it  is 
totally  reflected  out  of  the  crystal  in  the  direction  HX\  while 
the  extraordinary  ray  striking  the  balsam  at  a  less  oblique 
angle,  most  of  it  is  transmitted  through  the  crystal,  and  it 
emerges  at  the  opposite  side  to  which  it  entered,  K  This 
preparation  of  the  calcium  carbonate  is  termed  NicoPs  prisms. 
The  polariscope  of  the  microscope  consists  of  two  of  these 
prisms :  one  is  called  the  polarizer,  which  is  placed  beneath  the 
object  on  the  microscope,  and  the  other  is  called  an  analyzer, 
which  is  placed  above  the  objective  or  the  ocular.  They  are 
properly  mounted  in  tubes,  as  is  shown  in  the  illustration 

1  The  critical  angle  of  Canada  balsam  is  69.5°,  and  when  a  ray  of  light  strikes 
it  at  a  greater  angle  the  light  is  totally  reflected. 


254  BIOLOGICAL   LABORATORY  METHODS 

(Fig.  121),  so  that  they  will  fit  the  microscope  in  the  position 
desired.  When  the  light  passes  through  the  first  prism,  it  is 
reduced,  as  above  stated,  into  two  planes,  one  called  the  ordi- 
nary ray  and  the  other  the  extraordinary  ray ;  and  when  they 
strike'  the  film  of  balsam  separating  the  prisms,  the  ordinary 
ray  is  totally  reflected,  while  the  extraordinary  ray  passes  through 
the  second  prism  and  produces  the  effect  called  polarization. 
All  light  must  be  excluded  from  the  stage  of  the  microscope 
except  that  which  comes  through  the  polarizer,  because,  if  this 
precaution  is  not  taken,  the  field  will  not  be  sufficiently  darkened 
for  satisfactory  results.  When  the  polarizer  and  analyzer  are 


FIG.  I2i.  —  Zeiss'  Polarizer. 

in  their  places  on  the  microscope,  it  will  be  found  that  by  turn- 
ing the  analyzer,  there  is  a  position  when  the  largest  quantity 
of  the  light  reaches  the  eye,  and  also  a  position  when  the  light 
is  entirely  cut  off.  The -first  position  is  when  the  prisms  are 
parallel  to  each  other,  and  the  latter  when  they  are  almost  at 
right  angles. 

If  an  object  is  now  placed  on  the  stage  of  the  microscope 
when  the  polariscope  is  in  position,  the  object  remains  invisible 
when  the  analyzer  and  polarizer  are  at  right  angles  to  each 
other.  But  if  the  object  has  polariscopic  properties,  it  will 
become  partly  visible,  because  it  will  so  affect  the  beam  of  light 
passing  through  the  first  prism  as  to  polarize  the  light  again, 
and  the  object  will  be  partially  visible  in  the  analyzer,  since  the 


POLARIZATION   OF  LIGHT  255 

polarizer  ray  is  changed  to  another  plane  by  the  interposed 
object.  These  objects  are  called  polariscopic —  such  as  thin  films 
of  selenite,  certain  woody  tissues  of  plants,  starch  grains,  sec- 
tions of  horn  ;  some  animal  and  plant  hairs  belong  to  this  class. 
Beautiful  colors  become  visible  in  the  microscope  when  these 
polariscopic  objects  are  interposed  between  the  polarizer  and 
analyzer.  To  have  these  color  effects  the  object  must  not  be 
too  thick  or  too  thin.  If  the  substance  is  quite  thick  the  light 
will  assume  its  white  condition,  and  if  too  thin  a  gray  result  is 
secured  on  a  black  ground,  so  that  an  effort  must  be  made  to 
obtain  intermediate  thicknesses. 

Adjustment  of  the  polariscope.  —  Swing  out  the  reflector  and 
the  Abbe  illuminator  and  take  out  the  diaphragm.  Insert  the 
polariscope  beneath  the  stage  in  the  position  occupied  by  the 
diaphragm.  Place  back  the  illuminating  apparatus.  Put 
the  analyzer  above  the  ocular.  Now  shade  the  stage  so  that  no 
ray  of  light  can  reach  it  except  that  which  is  reflected  through 
from  the  Abbe  illuminator  beneath.  The  analyzer  must  be 
adjusted  to  the  ocular  by  raising  or  lowering  until  the  field 
becomes  clear  and  sharp  to  the  outer  edge.  Now  turn  the 
analyzer  until  the  brightest  amount  of  light  comes  through  from 
the  stage  and  determine  this  relative  position  of  the  analyzer 
and  polarizer.  Then  turn  the  analyzer  so  that  all  light  is 
excluded  and  mark  this  position.  Upon  examination  it  will  be 
found  that  these  two  positions  are  90°  apart.  When  the  greatest 
amount  of  light  is  transmitted  to  the  eye  through  the  analyzer 
we  see  the  extraordinary  ray,  and  this  is  the  position  for  observ- 
ing the  image  of  the  object  it  is  desired  to  examine  under  the 
influence  of  the  polarized  ray. 

The  polariscope  is  used  to  answer  among  others  the  following 
questions  :  (i)  Is  an  object  simply  refractive  in  its  properties, 
that  is  optically  homogeneous  ?  (2)  Has  the  object  the  power  of 
double  refraction  ?  (3)  Are  there  minute  crystals  in  the  section 
which  are  not  visible  under  the  microscope  with  the  use  of  the 
ordinary  white  light  unpolarized  ?  (4)  Is  it  possible  to  distinguish 
the  crystals  in  rock  sections  and  determine  the  composition  of  the 


256  BIOLOGICAL   LABORATORY   METHODS 

rock  ?  (5)  Are  the  beautiful  and  striking  effects  produced  by  the 
following  objects  characteristic  :  starch,  fibres  of  plants,  hairs  of 
animals  and  plants,  cellular  tissue,  muscular  tissue  of  animals, 
sections  of  bone,  hoofs,  horn  ? 

Selenite  should  comprise  part  of  the  polariscope  outfit  because 
it  is  useful  at  times  in  increasing  the  thickness  of  certain  sec- 
tions which  are  too  thin  to  produce  the  color  effects.  The 
selenite  may  be  introduced  at  any  position  between  the  polarizer 
and  the  analyzer.  This  selenite  stage,  as  it  is  sometimes  called, 
also  greatly  aids  in  the  study  of  substances  which  have  the 
power  of  only  single  refraction,  or  mono-refringent,  such  as  glass 
which  has  been  strained,  and  other  substances  which  have  not 
the  correct  thickness  to  cause  polarization.  In  such  cases  the 
selenite  produces  a  colored  field  in  which  the  crystals  are  brought 
out  clear  and  sharp. 

Mohl  has  done  much  valuable  work  with  the  polarizers  in 
investigating  the  effects  of  the  light  on  vegetable  organisms,  and 
Messrs.  Griffith  and  Henfrey  thus  sum  up  the  results  secured  by 
him:1- 

"  It  is  easy  to  ascertain  whether  an  organic  body  shows  posi- 
tive or  negative  colors,  by  comparing  its  color,  when  seen  with  a 
plate  of  gypsum  in  a  certain  definite  position,  with  the  color 
given  under  the  same  circumstances  by  a  strip  of  glass  brought 
into  a  state  of  tension  by  a  slight  bending,  or  with  the  colors  of 
a  suddenly  cooled  globule  of  glass.  In  this  way  the  author 
determined  that  the  fibres  of  a  spiral  vessel  displayed  negative 
colors,  and  the  laminae  of  a  starch  corpuscle  positive  colors,  and 
then  applied  these  organic  structures,  by  comparison,  for  ascer- 
taining the  properties  of  other  objects.  The  objects  to  be 
examined  should  be  mounted  in  a  liquid  or  other  substance, 
rendering  them  as  transparent  as  possible,  such  as  glycerin, 
Canada  balsam,  or  an  essential  oil. 

"  When  ordinary  globular  or  cylindrical  cellular  tissues  are 
viewed  by  cross-sections,  their  substance  is  seen  to  be  doubly 
refractive ;  for  when  the  prisms  cross,  the  circular  sections  of 

1  "  Micrographic  Dictionary,"  Griffith  and  Henfrey,  pp.  612,  613. 


POLARIZATION   OF   LIGHT  257 

cell  walls  appear  like  rings  of  bright  light  on  a  black  ground, 
but  with  the  ring  divided  into  four  quadrants  by  dark  stripes,  as 
if  a  black  cross  lay  over  it ;  when  the  prisms  are  placed  parallel, 
the  parts  of  the  section  previously  bright  appear  dark,  and  vice 
versa,  on  a  bright  field.  If  a  section  of  polyhedral  cellular 
tissue  is  viewed  in  the  same  way,  the  appearances  are  somewhat 
different,  since  the  cut  edges  are  here  straight  lines,  variously 
inclined  toward  the  prisms  ;  those  which  are  perpendicular  to 
the  prisms  are  invisible,  while  those  standing  obliquely  are 
bright  in  their  whole  length.  In  general,  cell  membrane  acts 
more  powerfully  on  the  light  the  denser  its  substance,  and  soft 
collenchymatous  tissues  are  far  less  powerfully  doubly  refractive 
than  wood  cells.  When  the  cells  have  walls,  much  thickened, 
it  is  common  for  the  primary  cell  membrane  to  be  much  more 
powerfully  refractive  than  the  secondary  layers. 


"  Let  us  suppose  that  between  the  lower  prism  and  the  object 
is  placed  a  plate  of  selenite,  giving  a  red  field ;  the  plate  is  then 
rotated  so  that  its  neutral  axes  are  at  an  angle  of  45°  with  the 
prisms.  A  section  of  a  cylindrical  vegetable  cell  will  be  seen 
to  be  divided  into  four  quadrants :  the  two  alternate  quadrants, 
whose  middle  lines  correspond  to  the  neutral  axes  of  selenite, 
are  either  blue  or  green,  the  other  two  yellow  or  red ;  if  the 
selenite  is  then  rotated  so  that  its  neutral  axes  are  perpendicular 
to  the  prisms,  the  colors  will  be  all  lost ;  but  on  continuing  the 
rotation,  they  appear  in  the  reverse  order  —  what  was  blue 
appearing  yellow,  and  vice  versa.  When  the  walls  are  rectilinear, 
all  the  cell  walls  perpendicular  to  one  of  the  prisms  will  give  the 
color  of  the  field,  all  those  which  run  parallel  with  one  of  the 
neutral  axes  of  the  selenite  plate,  or  form  no  great  angle  with  it, 
will  be  blue,  those  parallel  with  the  other  axis  yellow. 

"It  is  found  that  the  vegetable  structures  fall  into  two  classes 
in  reference  to  these  colors,  in  one  of  which  classes  all  layers 
lying  obliquely  in  the  direction  of  a  right-wound  screw  are  tinged 
blue  and  yellow,  those  oblique  in  the  opposite  direction  yellow 


258  BIOLOGICAL   LABORATORY  METHODS 

or  red ;  in  the  other  class,  the  colors  under  the  same  conditions 
are  just  the  reverse  ;  so  that  one  class  are  optically  positive,  the 
other  optically  negative. 

"  The  optically  negative  are  the  ordinary  cell-membranes  of 
the  internal  organs  of  plants,  whether  in  their  natural  condition 
or  cellulose,  purified  by  the  help  of  nitric  acid  and  chlorate  of 
potash.  Collenchyma,  horny  endosperm-cells,  the  gelatinous 
cells  of  Algae,  etc.,  all  agree  in  this  property.  Optically  positive 
colors  are  given  by  cell  membranes  of  periderm  and  cuticular 
layers  of  epidermal  cells.  The  contrast  of  the  positive  and 
negative  colors  of  the  cuticle  and  other,  parts  of  the  cell  wall  is 
well  seen  in  the  epidermis  of  Aloe.  The  diversity  of  coloring 
under  polarized  light  here  corresponds  to  the  diverse  behavior 
under  treatment  with  iodine  after  maceration  in  solution  of 
potash  (secondary  deposits). 

"  The  longitudinal  sections  of  all  behave  like  the  cross-sec- 
tions ;  but  the  appearances  are  not  so  clear.  When  side  views 
of  the  surface  of  cells  are  obtained,  the  phenomena  are  very 
varied ;  but  these  are  not  best  seen  in  vessels  or  ducts  when  the 
thickening  layers  are  in  the  form  of  spiral  bands.  Thus,  if  one 
of  the  spiral  vessels  of  Musa  is  placed  (its  spiral  somewhat 
drawn  apart)  with  its  long  axis  perpendicular  to  one  of  the 
prisms,  the  fibres  on  the  upper  side  turn  to  the  left,  those  on  the 
under  side  toward  the  right;  and  when  the  selenite  plate  is 
interposed,  they  exhibit  the  complementary  colors.  When  the 
side  walls  of  cells  have  obscure  striation,  as  in  the  cells  of  coni- 
fers, the  liber  cells  of  Apocyneae,  etc.,  the  membrane  gives  evi- 
dence of  its  fibrillar  structure  by  the  yellow  or  blue  color 
developed  with  the  selenite  plate.  If  fibres  of  a  spiral  vessel 
cross  at  right  angles,  and  they  are  pressed  together,  they  neu- 
tralize one  another  when  they  cross :  when  the  prisms  are  used 
alone,  the  crossing  points  are  black,  the  rest  of  the  fibres  white ; 
when  the  selenite  plate  is  interposed,  the  crossing  points  exhibit 
the  color  of  the  field,  and  the  uncrossed  portions  of  the  fibres 
are  blue  or  yellow,  according  to  position. 

"  The  vicinity  of  a  round  bordered  pit,  as  in  the  wood  cells  of 


POLARIZATION   OF   LIGHT  259 

Firms,  exhibits  a  black  cross  when  seen  perpendicularly  by 
polarized  light.  The  black  cross  and  the  colors  exhibited  by 
starch  are  well  known.  Chlorophyl  does  not  seem  to  act  on 
polarized  light,  nor  the  primordial  utricle  of  cells,  except  a  trace 
when  contracted  by  weak  alcohol. 

"  The  polarization  apparatus  is  exceedingly  useful  for  the 
detection  of  crystals  (Raphides)  in  vegetable  tissues,  when  they 
are  so  small  as  to  be  readily  overlooked ;  and  the  larger  kinds 
form  beautiful  objects  with,  and  often  without,  the  selenite 
plate." 

By  an  elaboration  of  these  experiments  with  sections  of  vari- 
ous kinds  of  plants,  much  useful  information  will  be  gleaned, 
which  will  enable  the  microscopist  to  arrive  at  conclusions  in  no 
other  way  attainable.  The  limit  and  the  character  of  this  work 
will  not  permit  of  more  extended  discussion  of  this  interesting 
subject,  but  the  director  of  the  laboratory  will  give  the  students 
much  valuable,  as  well  as  interesting,  information,  if  he  will 
require  them  to  make  frequent  tests  with  the  polariscope,  while 
studying  cross-sections  of  the  plants.  For  a  full  account  of 
polarization  in  microscopical  investigation,  the  student  is  referred 
to  the  writings  of  Dippel,  Nageli,  and  Schwendener. 


CHAPTER   XVII 

USEFUL    FORMULAE    AND    TABLES 

THERE  are  many  useful  formulae  and  tables  which  are  of 
importance  to  microscopists,  scattered  through  the  pages  of 
journals  and  books  devoted  to  biological  research,  and  their 
enumeration  would  much  more  than  fill  the  leaves  of  a  book 
larger  than  this.  The  attempt,  therefore,  will  be  made  to  men- 
tion only  a  few  of  the  more  important :  — 

Cleaning  glass.  —  i.  Place  in  a  wide-mouth  bottle  a  strong 
solution  of  borax  and  washing-soda,  or  caustic  potash.  The 
glasses  placed  in  this  will  soon  become  cleaned  of  gums  and 
other  foreign  matters. 

2.  Dichromate  of  potash 10  grammes 

Hot  water 50  cc. 

After  cooling,  add  slowly  sulphuric  acid         .         .         50  cc. 

First  remove  the  covers  from  slides  by  heating  two  or  three 
seconds  over  Bunsen's  burner ;  place  the  glasses  in  a  porcelain 
dish,  pour  over  them  the  fluid,  and  heat  on  water-bath  for  ten 
minutes.  When  clean,  wash  the  glasses  and  immerse  in  solution 
of  dilute  caustic  soda,  warm  for  five  minutes,  place  in  spirits, 
and  dry. 

3.  "  One  box  or  pound  of  crude   potash   dissolved   in  one 
gallon  of  water.     Bring  to  a  boil.     Dip  the  old  gelatin  plates 
in  this,  in  a  dipping-rack  that  will  keep  them  apart.     Dip  with 
movement,  and  when  the  film  is  gone,  remove  the  rack  and  glass 
into  another  vessel  of  warm  water,  and  then  wash  well  in  run- 
ning water.     Take  them,  one  at  a  time,  and  rub  with  rag  covered 
with  pulverized  cuttlefish  bone.     Put  them  in  a  rack  to  dry  and 
then  pack  away  for  use.     When  the  plates  are  to  be  used,  polish 

260 


USEFUL   FORMULA   AND  TABLES  26 1 

off  the  fishbone  and  dust  with  a  piece  of  soft  linen."     (HOLMES 
AND  GRISWOLD.) 

4.  Dr.  F.  Knauer's  method  :    The  slides  are  placed  in  a  vessel 
containing  about  half  a  liter  of  10  per  cent  solution  of  lysol,  and 
boiled  for  twenty  to  thirty  minutes.     The  still  seething  vessel  is 
then  placed  under  the  faucet  and  the  water  allowed  to  flow  in 
until  the  solution  becomes  quite  clear,  when  the  slides  are  taken 
out  and  dried. 

5.  A  vessel  containing  a  lump  of  washing-soda,  or  "Gold 
Dust"    washing-powder,    and    water    brought    to    boiling,    will 
remove  all  the  gum  from  slides  and  cover-glasses,  so  that  the 
latter  are  easily  recovered. 

6.  Place  in  a  wide-mouth  jar  a  mixture  of  benzin,  turpentine, 
and  benzol,  and  let  the  slides  remain  in  this  all  night.     Take 
out  and  polish  with  a  soft  cloth.     (F.  L.  JAMES.) 

Cements  and  Mounting  Media 

7.  Canada  balsam:  Heat  the  solid  balsam  until  brittle  when 
cold,  and  then  dissolve  in  either  benzol,  xylol,  chloroform,  or 
turpentine  to  the  required  consistency.     The  xylol  solution  is 
generally  considered  to  be  the  best,  but  it  sets  more  slowly  than 
the  benzol  solution. 

8.  Damar :  Dissolve  without  heating  in  xylol,  or  one  of  the 
other  menstrua  mentioned  in  (7). 

9.  To  prepare  glycerin  jelly  for  delicate  mounting :  Dissolve 
transparent  isinglass  in  a  sufficient  quantity  of  distilled  water  to 
make  a  stiff  jelly.     When  at  ordinary  temperature  of  working 
room  add  one-tenth  as  much  good  glycerin  and  a  little  solution 
of   borax,  carbolic    acid,  or  camphor  water.     Filter  while  hot 
through  washed  muslin,  and  subsequently  add  a  little  alcohol 
to  improve  its  working.     (Amer.  Mic.Jour.,  1881.) 

10.  Alcohol-glycerin.     Especially  recommended   for   plants, 
entire  or  in  parts. 

Glycerin I  part 

Alcohol  (96  per  cent)      .         .         .         .     i  part 

Water i  part     (A.  B.  AUBERT.) 


262  BIOLOGICAL  LABORATORY   METHODS 

1 1 .  Glycerin  jelly :  — 

Glycerin 120  grammes 

Gelatin 30  grammes 

Water 60  grammes 

Dissolve  the  gelatin  in  warm  water,  add  the  glycerin,  filter. 
For  vegetable  and  animal  tissue.  (A.  B.  AUBERT.) 

12.  Farrant's  medium  for  delicate  plants  or  animal  tissue: 

Gum  arable 30  grammes 

Glycerin 30  grammes 

Arsenious  oxide o.i  gramme 

Water .     30  cc. 

To  be  kept  in  a  stoppered  bottle  with  a  lump  of  camphor. 

Fixing  and  Clearing  Formula 

13.  Mayer's  albumen  fixing  method  (Lee):  — 

White  of  egg 50  cc. 

Glycerin .     50  cc. 

Salicylate  of  soda  -. I  gramme 

Shake  well  together,  and  filter  into  a  clean  bottle. 

The  following  is  a  modification  of  Mayer's  method,  given  in 
the  Amer.  Mic.Jour.,  p.  119,  1895:  — 

"  A  layer  of  Mayer's  albumen  was  spread  on  the  slide,  and 
the  sections  arranged.  Then  a  wash  -|  per  cent  collodion  was 
spread  over  the  surface  evenly  with  a  camel's-hair  brush.  This 
is  allowed  to  dry,  which  takes  place  in  about  one  minute,  but  a 
longer  time  does  no  harm  ;  practically  one  slide  dries  while  the 
next  is  being  prepared.  During  the  drying  many  small  air 
bubbles  appear,  the  presence  of  which  indicates  the  right  degree 
of  dryness ;  these  do  not  cause  any  inconvenience,  as  they  dis- 
appear during  the  subsequent  processes.  When  dry  the  slide  is 
put  up  without  heating  into  a  jar  of  xylol  or  benzin  for  half  an 
hour  or  more  to  dissolve  the  paraffin.  A  stay  of  several  hours 
will  not  injure  the  tissues.  The  paraffin  may  be  removed  in 
three  to  five  minutes  by  constantly  moving  the  slide  in  the 
benzin.  The  benzin  or  xylol  is  removed  by  95  per  cent  alcohol, 
and  the  sections  are  stained  and  mounted  as  desired," 


USEFUL  FORMULA   AND   TABLES  263 

Preserving  Medium  for  Plants 

14.  Formalin  or  formol  preserving  medium:  — 

Formaldehyd 40  parts 

Water 100  parts 

Chlorophyl  and  all  delicate  parts  of  plants  are  well  preserved 
in  this  solution,  and  the  colors  are  not  destroyed  —  some  flowers 
are  preserved. 

Photographic  Developing  Solutions 

15.  Carbutt's  pyrogallic  acid  developer  :  — 

No.  I.     Pyrogallic  Acid  Stock  Solution 

300  cc.         distilled  or  ice  water 10  ounces 

1  gramme   oxalic  acid 15  grains 

2  grammes  bromide  potassium         .         .         .         .         -3°  grains 

Then  add  Schering's  pyrogallic  acid  i  ounce  (30  grammes), 
and  water  to  make  16  fluid  ounces  (480  cc.). 

No.  2.     Stock  Soda  Solution 

300  cc.  water 10  ounces 

1 20  grammes  soda  sulphite  crystals          ....  4  ounces 

60  grammes  soda  carb.  crys.  (or  dry  grain  I  ounce)       .  2  ounces 

30  grammes  potash  carbonate I  ounce 

Dissolve,  and  add  water  to  make  measure  16  fluid  ounces 

(480  cc.). 

No.  3.     Bromide  Solution 

14  grammes  bromide  of  sodium  or  potassium         .         .       \  ounce 
150  cc.  water 5  ounces 

Dilute  2  ounces  of  stock  No.  2  with  7  ounces  of  water  for  cold 
weather,  and  10  to  12  of  water  in  summer.  To  3  ounces  of 
dilute  No.  2  add  \\  to  2^  drachms  (6  to  10  cc.)  of  No.  i.  The 
more  pyro,  the  denser  the  negative,  and  vice  versa.  No  yellowing 
or  fogging  need  be  apprehended  if  our  directions  are  followed. 
Development  should  be  continued  until  the  image  seems  almost 
buried,  then  wash  and  place  in  fixing-bath. 


264  BIOLOGICAL   LABORATORY  METHODS 

For  instantaneous  exposures  take  for  a  5  x  8  or  6J  x  8 \  plate  3 
ounces  of  dilute  No.  2.  Lay  the  plate  to  soak  in  this,  and  cover 
pan.  Put  2  drachms  of  No.  i  into  the  graduate,  and  3  drops  of 
bromide  solution.  Pour  the  soda  solution  off  of  the  plate  into 
the  pyro  and  back  over  the  plate  ;  let  development  proceed,  and 
examine  occasionally.  Keep  solution  in  gentle  motion  over  the 
plate..  A  very  short  exposure  may  take  ten  minutes  to  fully 
develop.  If  the  image  is  not  fully  brought  out  this  time,  add 
to  developer  in  pan  three  times  its  bu"_k  of  water,  and  let  plate 
lie  in  it  covered  over  for  half  an  hour  or  more  if  necessary,  until 
full  development  is  attained,  then  wash  and  proceed  as  directed 
under  head  of  developer. 

Cramers  Pyrogallic  Developer 

1 6.  Alkaline  solution  :  — 

177.6  cc.  water 60  ounces 

142     grammes  carbonate  of  soda  crystals         .        .          .5  ounces 
284     grammes  sulphite  of  sodium  crystals       .  .10  ounces 

A  smaller  quantity  of  sulphite  will  produce  a  warm  tone,  a 
larger  quantity  a  gray  or  bluish  black  tone. 

The  alkaline  solution  must  be  kept  in  well-stoppered 
bottles. 

If  the  negative  shows  a  yellow  stain  make  a  fresh  solution,  or 
try  another  lot  of  sulphite  of  sodium. 

Pyro  solution  :  — 

Dissolve  i  drachm  sulphite  of  sodium  crystals  in  6  ounces  of 
distilled  water,  add  acetic  acid  until  the  solution  turns  blue  litmus- 
paper  red,  and  finally  add  i  ounce  of  pyrogallic  acid. 

For  the  developer  use  the  following  proportions  :  — 

3.7  cc.  pyrogallic  acid  solution  i  drachm 

29.5  cc.  alkaline  solution i  ounce 

59.2  cc.  tepid  water  (for  winter  use)   ...       2  ounces,  or 

88.8  to  148  cc.  cold  water  (for  summer  use)  .         .         3  to  5  ounces 

17.  Eastman's  pyrogallic  developer  (for  films) :  — 


USEFUL  FORMULAE  AND  TABLES 


265 


No.  i 

14.2  grammes  pyrogallic  acid  . 

1.2  cc.  nitrous  or  sulphurous  acid 

947     cc.  water        . 

No.  2 

170     grammes  sulphite  of  soda  crystals 
113.6  grammes  carbonate  soda  crystals    . 
947     cc.  water       . 

To  develop  take  :  — 

29.6  cc.  No.  i 

29.6  cc.  No.  2          . 
59.2  cc.  water  . 


|  ounce 
20  minims 
32  ounces 

6  ounces 
4  ounces 
32  ounces 


I  ounce 

1  ounce 

2  ounces 


18.    Eastman's  pyrogallic  developer  (for  plates):  — 

No.  i 


170     grammes  sulphite  of  soda  crystals  . 
28.4  grammes  pyrogallic  acid 
947     cc.  water      .... 

No.  2 

113.6  grammes  carbonate  of  soda  crystals 
947     cc.  water       .... 


6  ounces 

I  ounce 

32  ounces 


4  ounces 
32  ounces 


To  develop  take  :  — 

29.6  cc.  No.  i 

29.6  cc.  No.  2 

78.8  to  118  cc.  water 


i  ounce 
i  ounce 
3  to  4  ounces 


In  warm  weather  use  more  water ;  in  cold,  less. 
19.    Hammer's  pyrogallic  developer:  — 


Pure  water 

Sulphite  of  soda  crystals 

Carbonate  of  soda  crystals 


No.  I 


No.  2 


Pure  water 
Oxalic  acid 
Pyrogallic  acid 


900  cc. 
150  grammes 
75  grammes 


720  cc. 

I  gramme 
30  grammes 


266  BIOLOGICAL   LABORATORY   METHODS 

To  develop  take  :  — 

No.  i 30  cc. 

No.  2     . 15  cc. 

Pure  water 90  to  180  cc. 

More  water  may  be  used  in  warm  weather,  and  less  in  cold 
weather. 

20.  Seed's  pyrogallic  developer :  — 

A 

296     cc.  water 10  ounces 

14.9  grammes  sulphite  of  soda  crystals  \  ounce 

Add  enough  pure  acetic  acid  to  this  to  turn  blue  litmus-paper 
slightly  red,  then  add  pyrogallic  acid  i  ounce. 

B 

473.6  cc.  water 16  ounces 

1 13.6  grammes  sulphite  of  soda  crystals          .         .         .         4  ounces 

C 

473.6  cc.  water 16  ounces 

113.6  grammes  sal.  soda  crystals    .....         4  ounces 

To  develop  take  :  — 

14.8  cc.  A |  ounce 

29.6  cc.  B i  ounce 

29.6  cc.  C i  ounce 

236.6  cc.  water 8  ounces 

For  double-coated  plates  use  18  ounces  of  water. 

2 1 .  Stanley  pyrogallic  developer  :  — 

No.  I.     Alkaline  Solution 

2368  cc.  pure  water 80  ounces 

1 86  grammes  sulphite  of  soda  crystals          .         .         .         6  oz.  (Troy) 
1 86  grammes  carbonate  of  soda  crystals        .         .  6  ounces 

No.  2.     Pyrogallic  Solution 

2368     cc.  water 80  ounces 

i     gramme    sulphite  of  soda \  drachm 

28.4  grammes  pyrogallic  acid      .         .         ...         I  ounce 

For  use,  mix  NO.  i  and  No.  2  in  equal  parts. 


USEFUL  FORMULA  AND  TABLES 


267 


Hydrochinon  Developers 

22.    Carbutt's  developer  :  — 

A 

Warm  distilled  water 600  cc. 

Sulphite  of  soda  crystals 120  grammes 

Sulphuric  acid    .         .         .         .         .         .    :     .  4  cc. 

Hydrochinon 23^  grammes 


Bromide  of  potassium 
Water  to  make  up  to 


2  grammes 
960  cc. 


Carbonate  of  potash 
Carbonate  of  soda  crystals 
Water  to  make 


C.     Accelerator 


Caustic  soda 
Water 


60  grammes 
60  grammes 
960  cc. 


30  grammes 
300  cc. 


For  underexposure  a  few  drops  of  this  are  added  to  developer. 
D.     Restrainer 


Bromide  of  potassium 
Water 


14  grammes 
150  cc. 


For  developer  take 

A 

B 

Water 

For  instantaneous  exposures     . 

30  cc. 

30  cc. 

1  2O   CC. 

For  portraits     . 

30  cc. 

30  cc. 

150  cc. 

For  landscapes,  Sen.  20-27 

30  cc. 

15  cc. 

90  cc. 

Full  exposures,  Sen.  16-20     . 

30  cc. 

25  cc. 

1  2O   CC. 

For  lantern  slides     

30  cc. 

25  cc. 

120   CC. 

23.    Seed's  developer  :  — 

A 

28.4  grammes  hydrochinon 
142     grammes  sulphite  of  soda  crystals 
0.6  gramme    bromide  of  potassium  . 
1634     cc.  water  (ice  or  distilled) 


i  ounce 
5  ounces 
10  grains 
55  ounces 


268  BIOLOGICAL  LABORATORY   METHODS 

B 

1 1.6  grammes  caustic  potash        .         .         .         .         .180  grains 
296     cc.  water    .         .         .         .         .         .         .10  ounces 

For  the  developer  take  :  — 

n8.4cc.  A .         .        4  ounces 

14.8  cc.  B    .         . 1  ounce 

The  temperature  of  room  should  be  from  21°  to  24°  C. 

Eikonogen  Developer 

24.  Cramer's  developer  :  — 

No.  i 

1776     cc.  distilled  water     .         .         .  .60  ounces 

85.8  grammes  sulphite  of  soda  crystals      -.         .         .         3  ounces 

28.6  grammes  eikonogen I  ounce 

Boil  for  a  few  minutes.     After  cooling,  pour  into  a  bottle  and 
keep  well  corked. 

No.  2 
984     cc.  water    .......       40  ounces 

28.6  grammes  carbonate  of  potash  I  ounce 

For  the  developer,  use  of  solution  No.  i,  3  ounces;  solution 
No.  2,  i  ounce.  In  hot  weather  dilute  with  an  equal  quantity 
of  cold  water. 

25.  Eastman's  developer  :  — 

No.  i 

85.8  grammes  sulphite  of  soda  crystals       ...         3  ounces 

28.6  grammes  eikonogen  .         .  .         .         .         i  ounce 

1776     cc.  water  . 60  ounces 

No.  2 

85.8  grammes  carbonate  of  potash       ....         3  ounces 
888     cc.  water 30  ounces 

To  develop  take  :  — 

59.2  cc.  No.  I 2  ounces 

29.6  cc.  No.  2 i  ounce 

59.2  cc.  water 2  ounces 


USEFUL  FORMULA  AND   TABLES 


269 


Eikonogen-Hydroch  inon  Developers 

26.    Carbutt's    eikonogen    and    hydrochinon    developer   for 
orthochromatic  plates,  "  celluloid  "  films,  and  transparencies  :  — 


Distilled  water 
Sulphite  of  soda  crystals 
Eikonogen 
Hydrochinon     . 
Water  to  make  up  to 


600  cc. 
1 20  grammes 
22  grammes 
io|  grammes 
960  cc. 


Distilled  water  .         .         . 
Carbonate  of  potash  . 
Carbonate  of  soda  crystals 
Water  to  make  up  to 


600  cc. 

60  grammes 
60  grammes 
960  cc. 


DEVELOPER 


For  developer  take 

A 

B 

Water 

For  instantaneous  exposures    . 

30  cc. 

30  cc. 

120  CC. 

For  landscapes,  Sen.  20-27 

30  cc. 

15  cc. 

90  CC. 

Full  exposures,  Sen.  16—20    . 

30  cc. 

25  cc. 

120    CC. 

For  lantern  slides    ..... 

30  cc. 

25  cc. 

120    CC. 

Full  exposures       ....     and  2  to  6  drops  restrainer  D  to 

each  ounce  developer..    (See  below.) 


NOTE.  —  More  of  A  will  increase  density,  more  of  B  will  increase  detail 
and  softness.  Temperature  of  developer  should  not  vary  much  below  1 8°  C. 
nor  above  24°  C.  The  after  treatment  is  the  same  as  with  any  other  developer. 

27.    Seed's  eikonogen-hydrochinon  developer  :  — 

No.  i 

42.6  grammes  sulphite  of  soda l|  ounces 

1 6.8  grammes  eikonogen 240  grains 

3.9  grammes  hydrochinon 60  grains 

947     cc.             distilled  water 32  ounces 


2/0  BIOLOGICAL  LABORATORY   METHODS 

No.  2 

113.6  grammes  carbonate  of  soda          ....         4  ounces 
947     cc.  water 32  ounces 

To  develop  take :  — 

59.2  cc.  No.  I 2  ounces 

29.6  cc.  No.  2 I  ounce 

29.6  cc.  water I  ounce 

For  double-coated  plates  use  5  ounces  of  water. 

28.  Metol  developer  :  — 

Cramer's  Developer 

5.2  grammes  metol 80  grains 

85.2  grammes  sulphite  of  soda  crystals       ...  3  ounces 

28.4  grammes  carbonate  of  soda  crystals  I  ounce 

I     gramme    bromide  of  potassium  .         .         .         .  12  grains 

2368     cc.             water  . 80  ounces 

Used  for  developing  underexposed  plates. 

29.  Glycin  developer : 

Jules  Fuers  ?s  Formula 


Sulphite  of  soda  crystals 125  grammes 

Potassium  carbonate     .......       50  grammes 

Glycin          .         .         .         .         .         .         .  '   .       50  grammes 

Hot  water   .         .         .         .         .         .  .         .  1000  cc. 


Potassium  carbonate    .         .         /        .         .         .         .125  grammes 
Water          .         .         .         .         .  .         .  1000  cc. 


Sulphite  of  soda  crystals       .         .         .         .         .         .125  grammes 

Potassium  carbonate    ......  250  grammes 

Glycin .         .         .50  grammes 

Hot  water  .........  1000  cc. 

C  is  a  concentrated  one-solution  developer. 
For   normal  exposure,  take  i   part  of  A,  2  parts  of  B,  and 
i  part  of  water,  or  i  part  of  C  diluted  with  3  times  its  bulk  of 


USEFUL  FORMULA  AND  TABLES  2/1 

water,  or  i  to  6  for  underexposure.  For  overexposure,  3 
parts  of  A,  to  2  parts  of  B  and  3  parts  of  water.  For  under- 
exposure, developer  should  be  freely  diluted  to  give  time  for 
developer  to  act  on  slightest  light  impressions  without  "  plug- 
ging "  high  lights.  For  unknown  exposures,  take  i  part  of  B, 
2  parts  of  A,  and  2  parts  of  water,  to  which  add  15  drops  of 
bromide  of  potassium,  10  per  cent.  If  the  details  appear  in  less 
than  thirty  seconds,  the  plate  is  overexposed.  Any  tendency  to 
harshness  must  be  remedied  by  addition  of  more  potash  and 
further  dilution.  (International  Annual  for  1902.) 

Fixing-baths 

30.  Carbutt's  new  acid  fixing  and  clearing  bath  :  — 

Sulphuric  acid          .          .         .          .         .         .          .         4  cc. 

Hyposulphite  of  soda       ......     480  grammes 

Sulphite  of  soda       .         .         .         .  .         .60  grammes 

Chrome-alum x          .         .         .         .         .         .  30  grammes 

Warm  water    ........    1920  cc. 

Dissolve  the  hyposulphite  of  soda  in  1440  cc.  of  water,  the 
sulphite  of  soda  in  180  cc.  of  water,  mix  the  sulphuric  acid 
with  60  cc.  of  water,  and  pour  slowly  into  the  sulphite  of  soda 
solution,  and  add  to  the  hyposulphite  ;  then  dissolve  the  chrome- 
alum  in  240  cc,  of  water  and  add  to  the  bulk  of  solution,  and  the 
bath  is  ready.  This  fixing-bath  will  not  discolor  until  after  long 
usage,  and  both  clears  up  the  shadows  of  the  negative  and 
hardens  the  film  at  the  same  time. 

After  negative  is  cleared  of  all  appearance  of  silver  bromide, 
wash  in  running  water  for  not  less  than  half  an  hour,  to  free 
from  any  trace  of  hypo  solution.  Swab  the  surface  with  wad 
of  wet  cotton,  rinse,  and  place  in  rack  to  dry  spontaneously. 

3 1 .  Cramer's  fixing-bath  :  — 

No.  i 

908.8  grammes  hyposulphite  of  soda         ...         .32  ounces 
2841.6  cc.  water 96  ounces 

1  During  cold  weather  use  only  half  the  quantity  of  chrome  alum  in  above. 


2/2  BIOLOGICAL  LABORATORY   METHODS 

No.  2 

113.6  grammes  sulphite  of  soda  crystals          .         .         •  •  4  ounces 

14.8  cc.  '          sulphuric  acid ^  ounce 

56.8  grammes  powdered  chrome-alum          ...  2  ounces 

947     cc.            water 32  ounces 

Pour  No.  2  into  No.  i. 

32.  Hammer's  fixing-bath  :  — 

10     cc.  sulphuric  acid 3  drachms 

113.6  grammes  sulphite  of  soda         ....         4  ounces 
2960     ccv  water        ....         about     100  ounces 

When  this  is  about  half  dissolved,  add  2  pounds  of  hypo- 
sulphite of  soda;  after  the  hyposulphite  of  soda  is  dissolved, 
add  from  i  to  2  ounces  of  chrome-alum  dissolved  in  20  ounces 
of  water ;  then  add  enough  water  to  make  160  ounces. 

33.  Seed's  fixing-bath  :  — 

No.   i 

907     grammes  hyposulphite  of  soda         ...         2  pounds 

113.6  grammes  sulphite  of  soda  crystals    ...         4  ounces 

2841.6  cc.  water 96  ounces 

No.   2 

56.8  grammes  chrome-alum 2  ounces 

7.8  cc.  sulphuric  acid    .....         ^  ounce 

947     cc.  water         .         .         .        .         .        •     *  32  ounces 

Pour  No.  2  into  No.  i  while  stirring  rapidly. 

Intensification  Solutions  for  Photographic  Plates 

34.  Carbutt's  intensification  solution. 

With  correct  exposure  and  development,  intensification  need 
never  be  resorted  to.  The  following  formula  is,  however,  very 
effective,  and  the  most  permanent  of  all  methods  :  — 

No.  I 

1 6  grammes  bichlorid  of  mercury  ....     240  grains 

1 6  grammes  chlorid  of  ammonia     ....     240  grains 

600  cc.  distilled  water     .         .         .         .         .20  ounces 


USEFUL  FORMULA  AND  TABLES  2/3 

No.  2 

1 6  grammes  chlorid  of  ammonia     ....     240  grains 
600  cc.  water          ......       20  ounces 

Let  the  plate  to  be  intensified  wash  for  at  least  half  an  hour, 
then  lay  in  a  5  per  cent  solution  of  alum  for  ten  minutes,  and 
again  wash  thoroughly ;  this  is  to  insure  the  perfect  elimination 
of  the  hypo.  The  least  trace  of  yellowness  after  intensifying 
shows  that  the  washing  was  not  sufficient. 

Flow  sufficient  of  No.  i  over  the  negative  to  cover  it,  and 
allow  to  either  partially  or  entirely  whiten ;  the  longer  it  is 
allowed  to  act  the  more  intense  will  be  the  result.  Pour  off  into 
the  sink,  rinse,  and  flow  over  No.  2,  and  allow  to  act  one 
minute ;  wash  off,  and  pour  over  or  immerse  in  dilute  ammonia 
water  (i  drachm  strong  ammonia  in  8  ounces  of  water)  until 
changed  entirely  to  a  dark  brown  or  black.  Wash  thoroughly 
and  dry. 

35.  Cramer's  intensification  solution  :  — 

No.   i 

Bichlorid  of  mercury             .                  ...        saturated  solution 
Hydrochloric  acid,  to  each  ounce         .         .         i   drop 
Water .20  parts 

No.  2 

28.6  grammes  sulphite  of  soda I  ounce 

592     cc.  water 20  ounces 

Immerse  negative  in  first,  until  evenly  whitened,  then  rinse 
and  apply  the  sulphite  solution  until  the  negative  is  well 
blackened,  wash  and  dry. 

36.  Seed's  fixing-bath  :  — 

28.6  grammes  mercuric  chlorid        ....         I  ounce 

28.6  grammes  potassium  bromid  i  ounce 

1480     cc.  water 50  ounces 

Whiten  the  negative  in  this  solution,  wash  and  immerse  in 
equal  parts  of  a  saturated  solution  of  sulphite  of  soda  and  water. 
Wash  and  dry. 


2/4  BIOLOGICAL   LABORATORY   METHODS 

Reduction  Solutions 

37.  Carbutt's  reduction  solution.     (Farmer's  formula.) 

No.   i 

28.6  grammes  ferricyanid  of  potassium  I   ounce 

473     cc.  water 16  ounces 

No.  2 

28.6  grammes  hyposulphite  of  soda  I  ounce 

473     cc.  water .16  ounces 

Immerse  the  negative  in  the  hyposulphite  solution,  to  which 
have  been  added  a  few  drops  to  each  ounce  of  the  above  ferri- 
cyanid solution.  The  speed  of  reduction  depends  on  the  quan- 
tity of  ferricyanid  present.  Wash  thoroughly. 

38.  Cramer's  reduction  solution  :  — 

No.   i 

28.6  grammes  red  prussiate  of  potassium  i  ounce 

473     cc.  water          .         .         .         .  .16  ounces 

No.  2 

28.6  grammes  hyposulphite  of  soda  .         .         .         .         i  ounce 
473     cc.  water 16  ounces 

No.  i  is  affected  by  light  and  must  be  wrapped  with  opaque 
paper  and  kept  in  a  dark  place  when  not  in  use. 

Mix  8  ounces  of  No.  2  and  one  ounce  of  No.  i  and  use  in 
subdued  daylight.  Wash  thoroughly  to  eliminate  the  hypo- 
sulphite of  soda.  A  dry  negative  must  be  first  soaked  in  water, 
but  a  negative  just  fixed  may  be  transferred  at  once  to  the 
reducer  provided  all  of  the  hyposulphite  fixing-bath  has  been  well 
washed  out.  Keep  the  vessel  in  motion  while  reducing.  After 
washing  immerse  the  plate  in  a  solution  of 

572  grammes  sulphite  of  soda  crystals      ...         2  ounces 
592     cc.  water          .         .         ...         .         .20  ounces 

to  stop  the  action  of  the  reducer,  then  wash  thoroughly  in  run- 
ning water,  for  an  hour. 


USEFUL  FORMULA  AND  TABLES  2/5 

39.  Permanganate  of  potassium  reducer  :  — 

Permanganate  of  potassium      .....      0.5  gramme 

Sulphuric  acid          .         .  .         .         .         .          i  cc. 

Water 1000  cc. 

This  solution  keeps  for  a  long  time  and  may  be  applied  to  the 
negative  after  fixing,  even  if  the  same  has  not  been  thoroughly 
washed,  as  it  destroys  all  traces  of  fixing  soda  by  oxidation. 
During  the  process  the  tray  must  be  kept  in  motion.  (HENRY 
DIETRICH,  in  Anthony's  Photo.  BuL,  Vol.  XXXI.) 

40.  Persulphate  of  ammonia  reducer  :  — 

i     gramme  persulphate  of  ammonia       ....     15  grains 
28.4  cc.  water i  ounce 

When  the  desired  effect  is  produced  immerse  in  the  following, 
after  washing,  a  10  per  cent  solution  of  sulphite  of  soda;  wash 
and  dry.  Highly  indorsed  by  some  photographers.  (H.  S. 
LAUDER,  in  Wilson's  Photo.  Mag.,  Vol.  XXXVII.) 

Another :  — 

Persulphate  of  ammonia        .         .         .         .         .     2  to  3  per  cent. 
Glycerin  to  slightly  thicken. 

Another :  — 

.07  gramme  permanganate  potassium  i  grain 

113.6    cc.  water 4  ounces 

2    drops       sulphuric  acid 2  drops 

After  reduction  clear  with  2  drops  of  oxalic  acid  in  i  ounce 
of  water. 

41.  Monochromatic  light  filters  :  — 

Dr.  Nagel  in  Freiburg  gives  a  very  interesting  description 
of  how  to  make  monochromatic  filters  for  different  physiologi- 
cal purposes,  which  are  undoubtedly  of  importance  for  all 
ortho-chromatic  work,  the  three  color  processes,  and  all  color 
sensitive  plates.  The  several  color-filter  solutions  are  as  fol- 
lows :  — 

Orange  filter  for  the  spectrum  district  between  C  and  D.  — 
Prepare  a  solution  of  acetate  of  copper,  add  a  few  drops  of 


2/6  BIOLOGICAL  LABORATORY   METHODS 

acetic  acid,  and  then,  drop  by  drop,  a  concentrated  saffranin 
solution,  until  the  solution  will  admit  no  more  violet,  blue, 
green,  and  yellow  light. 

Yellow-ray  filter.  —  Add  to  a  saturated  acid  solution  of  ace- 
tate of  copper  a  saturated  acidified  solution,  "  Orange  G."  The 
liquid  has  a  brown  appearance,  and  passes  only  a  small  stripe 
of  yellow  light. 

Green-yellow  filter.  —  To  a  saturated  solution  of  bichromate 
of  potassium  acidified  with  acetic  acid  add  crystals  of  acetate 
of  copper,  and  heat  the  solution.  The  green  liquid  passes  only 
monochrome  green  light. 

Green-ray  filter.  —  To  a  saturated  solution  of  bichromate  of 
potassium  add  in  drops  a  solution  of  ammonia  carbonate  of  cop- 
per until  the  red  to  yellow  rays  are  absorbed.  To  this  solution 
add  a  few  drops  of  alkaline  fluorescent  solution. 

Blue-ray  filter.  —  A  weak  solution  of  methyl-green  is  mixed 
with  acetate  of  copper  solution  until  no  red  light  passes. 

Violet-ray  filter.  —  Ammonium  oxide  of  copper  solution  con- 
centrated and  mixed  with  7  parts  of  water  gives,  in  combination 
with  a  solution  of  permanganate  of  potash,  a  spectrum  which 
will  admit  only  the  light  between  F  and  G. 

Red  filter.  —  Dissolve  carmine  in  a  solution  of  carbonate  of 
lithium,  and  mix  the  solution  with  picric  acid.  (HENRY  DIETRICH 
in  Anthony's  Photo.  Bui,  Vol.  XXXI.) 

In  order  to  make  the  references  to  the  lines  C,  D,  F,  and  G 
mentioned  above  clear  the  following  table  is  given,  taken  from 
Roy.  'Mic.  Jour,  of  February,  1898  :  — 

Fraunhofer  Wave 

Lines  Lengths  (A.) 

A 759 

B 687 

C 656 

D  .           589 

E .527 

F 486 

G 43° 

H.             .       397 


USEFUL  FORMULA  AND  TABLES  2/7 

Distribution  of  colors  (after  Listing)  :  — 

X 

Red 723-647 

Orange 647-586 

Yellow 586-534 

Green 534~49* 

Bright  blue    .         .         .         .-        .         .         .         .         .  491-455 

Blue  violet     .........  455~424 

Violet    .  424-397 

42.    Printing  formulae  for  positives.     Carbutt's  vinco  :  — 
The  light  and  exposure.  —  Any  artificial  light  can  be  used  in 
an  ordinary  room,  turn  down  flame  of  gas  or  oil  light,  open  pack- 
age of  vinco,  place  piece  of  paper  over  negative  in  printing-frame 
(the  sensitive  side  may  be  known  by  its  tendency  to  curl  inwards), 
turn  up  light,  and  expose  for  an  average  good  negative  fifteen  to 
twenty  seconds  at  a  distance  of  twelve  inches  from  the  light. 
Metol-hydro  developer :  — 

20  ounces  water      .......  600  cc. 

30  grains  metol      .......  2  grammes 

IO  grains   hydrochinon   .          .          .          .                    .  f  gramme 

no  grains  sulphite  soda  gran.  .         ....  7  grammes 

no  grains  carbonate  soda  gran.        ....  7  grammes 

2  grains  potassium  bromide  .....  ^  gramme 

For  soft-detail  negatives,  shorten  exposure  and  use  full  strength ; 
for  vigorous  negatives,  dilute  one-half  with  water.  Lower  the 
flame  of  lamp  or  gas  to  about  one-half,  take  the  exposed  paper 
and  draw  through  the  developer  face  up,  thus  wetting  front  and 
back;  if  exposure  is  correct  development  will  proceed  uniformly, 
and  be  completed  in  thirty  to  sixty  seconds.  To  arrest  develop- 
ment place  at  once  in  following  solution  :  — 

Short  stop  and  hardener  :  — 

32  ounces  water        .......     960  cc. 

I  ounce   powdered  alum          .....       30  grammes 
i  ounce   table  salt  .         .         .         .          .         .       30  grammes 

Thirty  to  sixty  seconds'  immersion  is  sufficient ;  rinse  and  place 
in  fixing-bath. 


2/8  BIOLOGICAL   LABORATORY   METHODS 

32  ounces   water      .         .         .         .         .         .         .  960  cc. 

I  drachm  sulphuric  acid          .         .         .         .         .  4  cc. 

I  ounce     sulphite  of  soda  (dry)      ....  30  grammes 

8  ounces   hyposulphite  soda    .....  240  grammes 

Prints  should  remain  in  fixing  solution  not  less  than  ten 
minutes,  wash  in  running  water  for  thirty  minutes  to  one  hour, 
or  twelve  to  fifteen  changes  of  water.  After  well  rinsing  the 
prints  from  hypo  bath,  if  placed  in  the  alum  and  salt  bath  for 
five  minutes,  it  will  both  harden  the  surface  and  greatly  tend  to 
eliminate  the  hyposulphite  of  soda  and  shorten  time  of  washing. 

REMARKS. — To  make  ^ivote  flexible,  after  washing  immerse  for  a  few  min- 
utes in  one  to  ten  glycerin  and  water.  Bromide  in  developer  is  only  needed 
if  negatives  are  flat  and  lack  contrast,  then  a  few  drops  of  a  10  per  cent  solu- 
tion potassium  bromid  can  be  added  to  developer. 

CAUTION.  —  To  secure  success  from  the  start  with  vinco  hold  the  negative 
at  least  twelve  inches  from  gas  or  lamp  light,  and  if  a  Welsbach,  fifteen  to 
twenty  is  better.  Also  follow  our  instructions  as  to  using  a  weak  developer; 
overexposure  and  a  strong  developer  are  fatal  to  best  effects  with  vinco. 

To  stop  development  locally.  —  It  frequently  happens  that 
landscape  negatives  have  delicate  detail  in  the  shadows  ;  to  pre- 
serve them  in  the  print,  as  soon  as  the  image  is  out  far  enough 
in  the  shadows,  remove  from  developer,  rub  over  the  parts  with 
a  finger  dipped  in  the  short  stop,  or  with  a  tuft  of  cotton  wet 
with  same.  This  will  arrest  development  in  those  parts,  while 
allowing  the  rest  to  proceed. 

43.    Velox  developing-paper  :  — 

0.5  gramme  metol 7  grains 

14.2  grammes  sodium  sulphite  crystals  \  ounce 

2  grammes  hydrochinon 30  grains 

28  grammes  sodium  carbonate  crystals  .  .  .  400  grains 

296  cc.  water 10  ounces 

10  drops  bromide  of  potassium,  10  per  cent  about  10  drops 

Or, 

n8.4cc.  water 4  ounces 

14     grammes  sodium  sulphite  crystals         .         .         .     200  grains 

1.4  grammes  amidol 20  grains 

5     drops        10  per  cent  bromide  of  potassium 

solution about  5  drops 


USEFUL   FORMULAE  AND  TABLES  2 79 

If  blacks  are  greenish,  add  more  amidol ;  if  whites  are  grayish, 
add  more  bromide  of  potassium. 

44.  Fixing-bath :  — 

453.6  grammes  hyposulphite  of  soda  ....       1 6  ounces 
1894     cc.  water 64  ounces 

Then  add  the  following  hardening  solution  :  — 

148     cc.  water 5  ounces 

14     grammes  sodium  sulphite  crystals  ^  ounce 
88.8  cc.             commercial   acetic  acid   (25    per  cent 

pure  acid) 3  ounces 

14     grammes  powdered  alum  .         .         .         .  ^  ounce 

45.  Blue-prints :  — 

No.  i 

53.2  grammes  citrate  of  iron  and  ammonia          .         .         i|  ounces 
236.8  cc.  water    .  ' 8    ounces 

No.  2 

35.5  grammes  ferricyanid  of  potassium         .         .         .         i£  ounces 
236.8  cc.  water    ...!...         8    ounces 

Mix  equal  parts  of  No.  i  and  No.  2,  and  float  the  paper  on  the 
solution  for  three  minutes.  Plain  Rives  paper  should  be  used. 
Hang  up  to  dry  in  dark  room.  (International  Annual,  1902.) 

Prolonged  washing  will  clear  the  lights. 

Overexposure  may  be  corrected  by  washing  in  water  con- 
taining a  very  small  amount  of  ammonia — 10  drops  of  strong 
ammonia  to  i  pint  of  water  —  and  gradually  increasing  the 
strength  until  the  lights  are  cleared.  Dilute  solution  of  oxalic 
acid  will  work  the  same  results. 

The  colors  may  be  brightened  by  washing  in  water  with  a 
trace  of  hydrochloric  acid  or  citric  acid. 

Culture  Methods 

46.  Method  for  making  the  three  principal  media  based  on 
the  recommendation  of  the   Bacteriological  Committee  to  the 
American  Public  Health  Association.     Modified  by  H.  W.  Hill, 
Boston  Board  of  Health.     (Jour.  App.  Mic.,  Vol.  II,  p.  301.) 


280 


BIOLOGICAL   LABORATORY   METHODS 


Boil  30  grammes  thread  agar  in  I  liter  of  water  for  half  an  hour.     Make  up 
loss  by  evaporation  to  a  weight  of  1000  grammes.     Cool  and  solidify. 


Nutrient  Broth 

1.  Infuse      lean      meat 
twenty   hours   with    twice 
its  weight  of  distilled  water 
in    refrigerator,    say    1000 
grammes        meat,       2000 
grammes  water. 

2.  Make  up  weight  of 
meat  infusion  (and  meat) 
to  original  weight  by  add- 
ing   water,    i.e.    to    3000 
grammes. 

3.  Filter       infusion 
through    cloth   to   remove 
meat. 

4.  Titrate    and    record 
reaction  of  filtrate,  say  re- 
action -f  2.2  per  cent. 

5.  Weigh  infusion,  say 
1800  grammes. 

6.  Set  infusion  on  water- 
bath,  keeping  temperature 
below  60°  C. 

7.  Add  peptone,  I  per 
cent,  1 8  grammes;  add  salt, 
0.5  per  cent,  9  grammes. 

8.  After  ingredients  are 
titrated,  reaction  probably 
-f  2.3  to  +2.5. 

9.  Neutralize    (Fuller's 
method). 


Nutrient  Gelatin 
Ditto. 


Ditto. 

Ditto. 
Ditto. 

Ditto. 
Ditto. 


Nutrient  Agar 

Infuse  lean  meat  twenty 
hours  with  Us  own  weight 
of  distilled  water  in  refrig- 
erator, say  1000  grammes 
meat,  1000  grammes  water. 

Ditto,  i.e.  to  2000 
grammes. 


Ditto. 

Ditto,  say  reaction  +  4-2 
per  cent. 

Ditto,  say  900  grammes. 
Ditto. 


Ditto,    and    sheet       Add  peptone,  2  per  cent, 
gelatin,  10  per  cent,    18  grammes;    add  salt,  I 
per  cent,  9  grammes. 

Ditto;  reaction  probably 
+  4-5  to  +4-7- 


1 80  grammes. 

Ditto;  reaction 
probably  +  4.0  to 
+  5-0. 

Ditto. 


Ditto. 

To  the  900  grammes  of 
meat  infusion  (containing 
now  peptone  and  salt  also) 
add  900  grammes  of  the  3 
per  cent  agar  jelly  described 
at  head  of  this  column. 
Since  agar  is  neutral,  re- 
action is  unchanged. 


USEFUL   FORMULA  AND  TABLES 


28l 


10.  Heat  over  boiling  water  (or  steam)  bath  thirty  minutes. 

11.  Restore  weight  lost  by  evaporation  to  original  weight  of  filtered  meat 
infusion,  i.e.  that  on  which  the  percentage  of  peptone,  salt,  etc.,  were  calcu- 
lated, 1800  grammes  in  each  case. 

12.  Titrate,  reaction  probably  +0.3  to  +0.5. 

13.  Adjust  reaction  to  final  point  desired,  generally  to  +  1.5  per  cent. 

14.  Boil  five  minutes  over  free  flame,  with  stirring. 

15.  Add  water  if  necessary  to  make   up   loss  from  evaporation  to   1800 
grammes. 

1 6.  Filter  through  absorbent  cotton,  passing  the  filtrate  through  the  filter 
repeatedly  until  clear. 

17.  Titrate  to  determine  whether  or  not  the  desired  reaction  has  been 
maintained. 

1 8.  Tube  and  sterilize. 


47.   James  H.Wright's  method  of  anaerobic  cultivation  in  fluid 
media.     (Jour.  App.  Mic.,  Vol.  Ill,  p.  1006.) 

The  adjoining  figures  (122  and  123)  represent  a  simple  appa- 
ratus used  successfully  by  the  author  in  the  cultivation  of 
tetanus  bacillus.  The  apparatus  consists  of  a  simple  arrange- 
ment of  glass  and  rubber  tubes 
enclosed  in  an  ordinary  test-tube, 
with  a  plug  of  cotton  inserted  in 
its  mouth,  as  in  an  ordinary  cul- 
ture tube. 

When  it  is  desired  to  make  an 
anaerobic  culture,  the  culture  fluid 
is  put  in  the  test-tube,  and  inocu- 
lated in  the  usual  way.  By  suc- 
tion the  fluid  is  drawn  up  into 
the  system  of  tubes  to  a  level 
above  C,  after  which  the  rubber 
tube  E  is  compressed  with  the 
fingers,  and  the  tube  D  pushed 
downward  into  the  test-tube  in 
such  a  manner  as  to  fold  the  rub- 
ber tubes  D  and  C,  thus  render- 
ing the  fluid  4  perfectly  air-tight. 


FIG.  122.  FIG.  123. 

Wright's  Anaerobic  Apparatus. 


282  BIOLOGICAL  LABORATORY   METHODS 

48.  Preparation  of  potato  for  culture  of  bacteria  :  — 

Select  several  sound  potatoes,  and  boil  them  for  about  twenty 
or  thirty  minutes  in  a  vessel  of  water.  Pour  out  the  water.,  and 
allow  the  potatoes  to  cool.  Divide  them  with  a  sterilized  knife. 
The  inoculation  is  performed  in  the  usual  way,  after  being  as- 
sured that  the  potato  is  thoroughly  sterilized  before  the  inocula- 
tion is  done. 

Stains  and  Staining 

49.  Beale's  carmine  :  — 

Carmine  .         .         .         .         .         .         .  *       .       0.6  gramme 

Dissolved  in  boiling  solution  of  ammonia.  Let  stand  for  an 
hour  to  cool  and  to  permit  superfluous  ammonia  to  escape.  Add 
to  the  solution  :  — 

Water       .         .         .         .         .         .         .         .         .     60  cc. 

Glycerin  .         .         .         .         .         .         .         .         .60  grammes 

Absolute  alcohol        .         .         .         .         ...  15  grammes 

Permit  to  stand  for  some  time  and  filter. 

50.  Anilin    violet  (Hanstein's    "  Practical    Botany,"   Bower, 
P-  33  0:  "Dissolve  equal  parts  of  fuchsin  and  methyl-violet  in 
alcohol.     It  stains  cellulose  cell  walls  of  a  faint  violet  color,  and 
lignified  cell  walls  deep  violet.     It  is  especially  useful  for  bring- 
ing out  the  different  parts  of  the  bast,  since  the  bast-fibres  stain 
red,  whereas  the  sieve-tubes  and  the  parenchyma  scarcely  stain 
at  all.     The  protoplasm  is  stained  pink;    amyloid  substances, 
gums,  and  nuclei  stain  different  shades  of  red  ;  resins,  blue  ;  and 
tannin,  brick-red." 

51.  Differential    nucleolar    staining    (Gustave    Mann.      See 
Jour.  Roy.   Mic.  Soc.,  p.   690,    1891.):    "  Tissues,  both    vege- 
table  and   animal,  preferably  fixed  by  picro-corrosive   method 
(Mann's),  are  treated  for  ten  minutes  in  a  saturated  solution  of 
heliocin  in  50  per  cent  alcohol ;  the  sections  are  then  transferred 
for  from  five  to  fifteen  minutes  to  a  standard  watery  solution  of 
anilin  blue.      The  superfluous  stain  is   rapidly  washed  off  by 
distilled  water,  and  the  sections  placed  again  for  one  or  two 


USEFUL   FORMULAE   AND  TABLES  283 

minutes  in  the  heliocin  solution,  dehydrated,  cleared  by  resinified 
turpentine,  and  mounted  in  turpentine  balsam.  The  whole  of 
the  cell  and  the  nucleus  are  stained  blue,  the  nucleolus  red." 

52.  Staining   of    Chlorophyl    (Mann.      See  Jour.  Roy.  Mic. 
Soc.,  p.  689,  1891.):  "A  glass  vessel  is  filled  with  two  liters  of 
water,  to  which  six  drops  are  added  of  a  10  per  cent  solution 
of  cyanin  in  absolute  alcohol.     Then  a  small  quantity  of  either 
Spirogyra  jugalis  or  Spirogyra  nitida  is  placed  in  the  vessel,  which 
is  exposed  to  the  bright  daylight.     After  some  time,  varying  with 
the  temperature  of  the  room  and  the  activity  of  the  threads,  from 
three  to  twenty-four  hours,  the  whole  of  the  cyanin  will  have 
been  taken  up  by  the  threads.     The  ground-substance  of  the 
chlorophyl  bands  will  have  changed  from  a  green  to  a  bluish 
green  color,  while  the  oil  globules  and  many  of  the  microsomes 
between  the  bands  will  have  turned  blue,  showing  their  fatty 
nature." 

53.  Staining  cell  nuclei  of  pollen-grains  (Herr  A.  Meyer's 
method.     See  Jour.  Roy.  Mic.  Soc.,  p.  899,  1892.)  :  — 

Carmine 0.5  gramme 

Absolute  alcohol      . 20  ccm. 

Hydrochloric  acid    . 3°  drops 

Heat  for  thirty  minutes  in  water-bath  and  add 

Chloral  hydrate 25  grammes 

Filter  and  cool.     Stains  nuclei  of   pollen-grains  within  ten 
minutes  an  intense  red. 

54.  Staining  paraffin  sections  (Amer.  Mic.  Jour.,  XI,  p.  u, 
1890)  :  Dissolve  the  coloring  matter  in  absolute  alcohol,  and  drop 
the  solution  in  turpentine  until   the  desired  depth  is  secured. 
Sections  fixed  to  the  slip  by  the  collodion  method  are  placed  in 
the  oven  until  the  oil  of  cloves  is  completely  evaporated ;  the 
paraffin  is  dissolved  in  turpentine,  and  the  slide  is  then  put  in  the 
stain.     The  action  is  quick.     Overstaining  may  be  corrected  by 
placing  in  a  solution  made  with  equal  parts  of  acid,  free  absolute 
alcohol,  and  turpentine.    Meyer's  carmine,  methyl-green,  methyl- 
blue,  gentian  violet,  safranin,  Bismarck  brown,  eosin  fuchsin,  may 
be  used  as  indicated  with  good  results, 


284  BIOLOGICAL  LABORATORY   METHODS 

55.  Staining  of  protoplasts  and  cell  walls  (Herr  J.  af  Klercher, 
Jour.  Roy.  Mic.  Soc.,  p.   562,   1893,  and    Verhandl.  Biol.   Ver. 
Stockholm,  IV,  No.  14,  1892):     "If  the  object  examined  is  an 
aerial  part  of  a  plant,  the  oily  substances  are  first  removed  by 
ether   or    dilute    ammonia,   and,   after  washing   out  the   fixing 
material,  the  object  is  allowed  somewhat  to  dry  in  order  to  .pro- 
mote the  entrance  of  the  staining  substance.     But  if  it  is  only 
the  membrane  which  is  to  be  stained,  the  object  is  brought 
directly,  or  after  washing  out  the  fixing  material,  into  eau  de 
Javelle  or  eau  de  Labarracque,  and  left  there  till  all  the  proto- 
plasm is  dissolved.     After  careful  washing,  it  is  then   stained 
with  a  moderately  concentrated  solution  of  Congo  red,  and  finally, 
after  careful  washing,  placed  in  paraffin.     A  good  staining  of 
membranes  may  also  be  effected  by  successive  treatment  with 
iron  salts  and  potassium  ferrocyanid,  or  with  tannin  and  ferric 
chlorid." 

56.  Alum-carmine  (Boneval.     See  Amer.  Mic.  Jour.,  p.  78, 
1894.):- 

Ammonia  alum  .         .         .         .         .         .         .         I  to  5  grammes 

Carmine 4  grammes 

Distilled  water 100  grammes 

"  Boil  for  twenty  minutes,  taking  care  to  maintain  the  original 
volume  by  adding  water.  Filter  and  preserve  by  a  crystal  of 
thymol.  It  is  a  nuclear  stain  of  the  first  order ;  it  colors  admi- 
rably the  nuclei  of  tissues  fixed  by  osmic  acid,  which  makes  it 
valuable  in  many  instances  where  picro-carmine  is  worthless. 
The  color  is  well  preserved  in  glycerin.  Place  the  sections  for 
a  few  hours  in  a  vessel  containing  one  cc.  of  alum-carmine,  and 
wash  until  the  excess  of  color  is  removed.  This  stain  is  exceed- 
ingly penetrating,  so  that  tissues  may  be  colored  en  masse" 

57.  Rosanilin-violet   (Hanstein's   "Practical  Botany,"   Stras- 
burger,  p.  398) :    "  Equal   parts    of   methyl-violet   and   fuchsin 
(majenta),  mixed  and  dissolved  in  alcohol.     Shows  stratification 
of   cell  walls,  and   differentiates   sections  of   stems,  especially 
monocotyledons.     Stains  protoplasm  bluish  violet ;  amyloid  sub- 


USEFUL   FORMULA  AND  TABLES  285 

ASu/y/*        K^/ 

stances,  nucleus,  and  gums,  different  shades  of  red  ;  resins,  blue  ; 
tannin,  foxy  red  ;  cellulose,  pale  violet ;  lignin,  reddish  ;  bast- 
fibres,  deep  red ;  sieve-tubes  and  bast  parenchyma,  hardly  at  all." 

58.  Gram's  method  for  staining  Diphtheria  Bacilli :  Allow  the 
fixed  specimens  to  remain  for  20  to  30  minutes  in  an  anilin- 
water   solution   of   gentian   violet,    prepared    in   the    following 
manner :  — 

To  100  cc.  of  distilled  water  add,  drop  by  drop,  anilin  oil 
until  the  mixture  is  opaque.  Shake  well  after  each  addition  of 
anilin  oil.  Filter  through  moistened  filter-paper  until  perfectly 
clear.  To  100  cc.  of  the  filtrate  add  10  cc.  of  absolute  alcohol 
and  1 1  cc.  of  concentrated  alcoholic  solution  of  gentian  violet. 

After  remaining  the  required  time  in  this  mixture  the  speci- 
mens are  placed  for  about  five  minutes  in  the  following  iodin 
solution :  — 

Iodin I  gramme 

Potassium  iodid  . .2  grammes 

Distilled  water .          .    300  cc. 

This  solution  should  be  allowed  to  act  until  the  specimens  are 
black,  after  which  they  are  thoroughly  washed  in  alcohol,  which 
removes  the  black  color,  causing  the  specimens  to  appear  pale 
gray.  Dry  and  mount  in  balsam,  or  contrast  stain  with  carmin 
or  Bismarck  brown.  (Jour.  App.  Mic.,  Vol.  IV,  p.  1476.) 

59.  To  remove  anilin  stain  from  the  fingers  :  — 

a.  Use  successively  a  5  per  cent  solution  of  sodium  chlorid, 
feroxid  of  hydrogen,  and  lastly  alcohol.     (Jour.  Mic.  and  JVa:'. 
Set.,  Vol.  II,  p.  157.) 

b.  To  remove  silver  stains  from  fingers  :  — 

14.2  grammes  sulphate  of  sodium \  ounce 

7     grammes  chlorid  of  lime \  ounce 

29.6  cc.  water i  ounce 

c.  To  remove  pyrogallic  acid  from  hands.     Wash  with  a  10 
per  cent  solution  of  oxalic  acid,  or  sulphuric  acid  diluted  with 
water -i  -.20. 

d.  To  remove  developer  stains  from  fingers,  use  lemon  juice. 


286  BIOLOGICAL   LABORATORY   METHODS 

Miscellaneous  Recipes 

60.  Dead  black :  — 

Ivory  or  lampblack  2  grains,  a  drop  or  two  of  gold  size, 
thoroughly  mix  and  add  24  drops  of  spirits  of  turpentine.  Apply 
with  camePs-hair  brush  to  produce  a  fine  coating  of  black. 

61.  Black  polish  on  brass:  — 

1.  28.4  grammes  nitrate  of  silver         ....        i  ounce 
592     cc.  water .20  ounces 

2.  28.4  grammes  nitrate  of  copper  I  ounce 
592     cc.             water 20  ounces 

Mix  the  two  solutions  together  and  dip  the  brass  into  it  and 
heat  in  an  oven  until  the  degree  of  black  is  secured.  (National 
Druggist) 

Another  recipe  for  blackening  brass :  Clean  from  all  grease, 
cover  with  a  solution  of  nitrate  of  copper  and  apply  heat.  May 
be  lacquered  by  applying  shellac  varnish  and  gently  heating. 

62.  Dead-black  surface  on  brass  :    To  2  grains  of  lampblack 
add  just  enough  gold  size  as  will  hold  the  lampblack  and  mix 
well.     Add  drop  by  drop,  and  when  well  mixed  add  24  drops  of 
turpentine  and  stir  thoroughly.     Apply  with  camel's-hair  brush. 
(L.  A.  WILSON,  The  Mic,,  1897.) 

63.  Blackening  iron  and  steel:    Clean   thoroughly  and  im- 
merse in 

Mercuric  chlorid .  4  parts 

Cupric  chlorid 2  parts 

Hydrochloric  acid 12  parts 

Alcohol 10  parts 

Water '.  100  parts 

Dry  and  place  for  half  an  hour  in  boiling  water.  If  color 
is  not  deep  enough,  repeat  the  operation.  (Revue  Suisse) 

64.  To  keep  metallic  objects  from  rusting :  — 

White  wax 1 1  parts 

Suet 2  parts 

Dissolve  in  spirits  of  turpentine. 


USEFUL  FORMULA  AND  TABLES  287 

65.  Ink  for  writing  on  glass  :  — 

Brown  shellac 20  grammes 

Alcohol 150  cc. 

Dissolve  and  then  add  drop  by  drop  a  solution  of 

Borax        i 35  grammes 

Distilled  water 250  cc. 

If  this  precipitates  the  shellac,  add  more  alcohol  and  allow  the 
excess  to  evaporate.  One  gramme  of  methyl-blue  will  give  the 
color.  (Zeit.f.  angew.  Mikr.,  Bd.  V.) 

66.  Etching  fluid  for  glass  :  — 

1.  Dissolve  sodium  fluorite 36  grammes 

in  distilled  water 500  cc. 

and  add  potassium  sulphate     ....         7  grammes 

2.  Dissolve  zinc  chlorid 14  grammes 

in  distilled  water 500  cc. 

and  add  concentrated  hydrochloric  acid  .       65  grammes 

These  solutions  can  be  kept  in  ordinary  glass  vessels.  When 
wanted  for  use  equal  volumes  are  mixed  in  a  hollow  paraffin  cube. 
A  small  quantity  of  India  ink  added  will  enable  the  writer  to 
see  what  he  is  doing.  (Cent.  Zeit.f.  Opti.  u.  Mech.,  XII,  p.  57.) 

67.  Another  etching  fluid:   Mix  10  parts  of  fluorid  of  am- 
monia with  40  parts  of  sulphate  of  barium,  and  add  sulphuric 
acid.     The  salts  are  previously  well  mixed  in  a  leaden  vessel. 
The  following  method  is  also  good :   equal  parts  of  fluorid  of 
ammonia  and  sulphate  of  barium  are  dissolved  in  fluoric  acid. 
The  writing  is  done  with  an  ordinary  steel  pen.     After  half  an 
hour  the  writing   surface  is  washed    with    water.       (Anthony's 
Photo.  Bull,  Vol.  XXXI,  p.   120.) 

68.  Writing  on  glass  :  — 

Shellac 2  parts 

Venice  turpentine I  part 

Oil  of  turpentine 3  parts 

Lampblack I  part 

Dissolve  in  water-bath. 


288  BIOLOGICAL   LABORATORY  METHODS 

69.  Melt  together  spermaceti  four,  tallow  three,  and  wax  two 
parts ;  and  add  six  parts  of  either  red  lead,  white  lead,  or  Prus- 
sian blue,  according  to  color  desired.     The  mass  is  turned  out 
in  sticks  for  use.     (Popular  Science  JVews,  Formulae  or  Faber's.) 

70.  The  so-called  diamond  ink,  for  writing  on  glass,  consists 
of  a  mixture  of  fluoric  acid  and  barium.     The  latter  is  used  as 
a  body  for  the  acid.     Permit  to  remain  on  the  glass  for  fifteen 
minutes  and  then  rub  off  the  barium,  and  the  characters  will  be 
impressed  in  the  glass. 

71.  The  emission  of  pollen-tubes:  Dr.  B.  D.  Halsted  recom- 
mends that  pollen-grains  be  placed  in  a  10  to  70  per  cent  solution  of 
sugar,  and  the  pollen-grains  will  produce  tubes  within  a  few  hours. 

72.  Absolute  alcohol:  Add  anhydrous  cupric  sulphate  to  95 
per  cent  alcohol.     The  pulverized  cupric  sulphate  is  first  heated 
to  red  heat  to  drive  off  the  water  of  crystallization,  and,  after 
cooling,  is  shaken  in  the  alcohol  and  the  mixture    allowed  to 
stand  a  day,  the  liquid  decanted,  fresh  cupric  sulphate  added, 
and   the   operation    repeated   until  the   blue  color  due  to  the 
presence  of  water  is  almost,  if  not  quite,  destroyed.     To  test, 
treat  a  drop  of  the  alcohol  with  a  drop  of  turpentine  on  a  slide 
and  examine  under  the  microscope  to  detect  particles  of  water. 

73.  Softening  dried  material :  — 

Caustic  potash  ...  .     i    part 

Glycerin  .         . 5!  parts 

Water 5    parts 

74.  Schultze's    macerating   fluid :    This  is  used  for  separat- 
ing the  parts  of  woody  tissue  and  is  prepared  by  dissolving  in 
50  cc.  of  nitric  acid  i  gramme  of  potassium  chlorate.     Used  in 
small   quantities,  and  metallic    instruments  must  be  protected 
from  its  effects. 

75.  To  clean  the  hands  of  cements  :  — 

Castile  soap,  shaved  fine 15  parts 

Alcohol,  95  per  cent 10  parts 

Benzol,  ordinary 10  parts 

Ammonia  water 5  parts 

Glycerin 5  parts 


USEFUL  FORMULA  AND  TABLES  289 

Dissolve  the  soap  in  the  alcohol,  add  the  ammonia  and  benzol, 
and  after  thorough  agitation  the  glycerin.  After  using  the  soap, 
wash  the  hands  and  finish  by  rubbing  over  them  a  few  drops  of 
glycerin. 

76.  Solution  for  silvering  glass  (Burton's)  :  — 

1.  1.8  grammes  nitrate  of  silver    .         .         .         .         .  25  grains 
29.6  cc.             distilled  water I  ounce 

2.  1.8  grammes  potash  C.  P 25  grains 

29.8  cc.            distilled  water I  ounce 

A 

Equal  parts  of  solution  i  and  2.  Ammonia  to  just  dissolve 
precipitate  ;  solution  i  to  just  cause  coloration. 

B 

189     grammes  loaf  sugar                                  ^  2700  grains 

592     cc.             distilled  water        ....  20  ounces 

7.4  cc.             nitric  acid 2  drachms 

296     cc.             alcohol  (strong)    ....  10  ounces 

2368     cc.             distilled  water  to  make  up  to        .  80  ounces 

For  use : — 

29.6  cc.  Solution  A I  ounce 

29.6  cc.  Solution  B I  ounce 

Solution  B  improves  with  age,  while  A  deteriorates. 

77.  Silvering  glass  (by  M.  M.  Auguste  and  Louis  Lumiere)  : 
"  To  100  cc.  of  a  10  per  cent  solution  of  silver  nitrate,  ammonia 
is  added  drop  by  drop  until  the  precipitate  formed  is  redissolved. 
Too  much  ammonia  must  not  be  added  at  first,  for  this  might 
prevent  the  formation  of  the  precipitate.     The  volume  of  the 
solution  is  increased  to  a  liter  by  the  addition  of  distilled  water. 
This  is  solution  A.     Solution  B  is  made  by  diluting  commercial 
formaldehyde  of  40  per  cent  with  distilled  water  so  as  to  form 
a  i  per  cent  solution.     Solution  B  can  be  kept  for  some  time. 
Two  volumes  of  A  are  rapidly  mixed  with  one  volume  of  B,  and 
the  mixture  is  rapidly  poured  over  the  glass  to  be  coated.     In 
five  or  six  minutes,  at  a  temperature  of  15°  to  19°  C.  all  silver  in 


2QO  BIOLOGICAL   LABORATORY   METHODS 

the  solution  is  deposited  in  a  brilliant  layer,  which  can  then  be 
washed  with  water.  (Jour,  de  Physique,  January,  1895,  and 
Amer.  Jour.  Sci.  June,  1895.) 

78.  Silvering  glass  with  formalin  :  Dissolve  i  gramme  nitrate 
of  silver  in   100  cc.  of  distilled  water,  and  carefully  add  am- 
monia until  the  precipitate  has  dissolved  again.     An  excess  of 
ammonia  should  be  avoided  as  much  as  possible.     Dilute  the 
solution   with   distilled   water  to   i    liter.     In  a  second  bottle 
dilute  25  cc.  formalin  solution  with  distilled  water  to  i  liter. 
The  glass  plate  to  be  silvered  is  carefully  cleaned  and  waxed 
around   the   edges,   and   it  is  then  laid  in  a  horizontal   posi- 
tion.    Two  parts  of  the  silver  solution  are  then  mixed  quickly 
with  i   part  of  the  formalin  solution,  and  the  mixture  poured 
upon  the  glass  plate.     After  ten  minutes  the  silver  will  have 
precipitated    upon   the  glass    plate,   when   the    liquid    may  be 
poured  off  and  the  mirror  is  ready  to  be  washed  and  cleaned 
under   the   faucet.     (Anthony's   Photo.   Bull,  Vol.    XXXI,    p. 
288.) 

79.  Ground-glass  varnish  :  — 

6.3  grammes  sandarac 90     grains 

1.4  grammes  mastic 20     grains 

59.2  cc.  ether 2     ounces 

14.8  to  44.4  cc.  benzol         .         .         .         .        \  to  ij  ounces 

The  proportion  of  the  benzol  determines  the  character  of  the 
surface. 

80.  To  clean  a  negative  stained  with  silver :    Rub  with  cot- 
ton moistened  with  a  solution  of  cyanid  of   potassium,  wash 
well  and  dry. 

81.  Size  of  photographic  glass  and  mounts  :  — 

Petite if  X    3|  inches 

One-ninth  plate .2X2^  inches 

One-sixth  plate 2f  X    3^  inches 

One-fourth  plate 3?  X    4i  inches 

Half  plate       .         .         .         .         .         4*-  X  5^  and  4*-  X    6\  inches 

Whole  plate  (4-4) 6|  X    8$  inches 

Extra  (4-4) 8    X  10    inches 


USEFUL  FORMULA  AND  TABLES  2QI 

82.  Fixing  microscopic  objects  to  the  glass  slip.     Warm  the 
glass  slip  (40°  to  50°  C.)  to  remove  all  trace  of  moisture,  apply  a 
drop  of  mixture  prepared  as  follows  :  — 

White  lac 15  grammes 

Absolute  alcohol 100  grammes 

Dissolve  in  water-bath  and  decant  off  clear  liquid. 

After  alcohol  is  evaporated  from  the  slip,  a  hard  trans- 
parent coating  is  left.  This  may  be  softened  by  a  drop  of  oil 
of  lavender.  Arrange  the  object  on  the  slip,  heat  over  the 
lamp  until  the  oil  of  lavender  is  evaporated  and  the  object  is 
left  firmly  attached.  (M.  J.  TEMPERE,  in  Micrographie  Prepa- 
rateur^) 

83.  Wickersheim's  solution  for  preserving  specimens  in  large 
quantities  :  — 

Alum .  100  grammes 

Saltpetre 12  grammes 

Potash 60  grammes 

Arsenious  oxid 20  grammes 

Boiled  water 3000  grammes 

84.  Virodtzeff 's  solution  for  preserving  large  specimens  :  — 

Glycerin 2160  grammes 

Water ' 1080  cc. 

Alcohol .  45  cc. 

Thymol 5  grammes 

85.  Kaiserling's   method  for  preserving  pathological   speci- 
mens.    It  is  claimed  for  this  method  that  the  natural  colors  of 
the  specimens  are  preserved  almost  exact.     Place  in  the  follow- 
ing solution,  for  3  to  5  days  :  — 

No.  i 

Formalin 40  parts 

Water ¥         .  200  parts 

Potassium  nitrate 3  parts 

Potassium  acetate 6  parts 


2Q2  BIOLOGICAL   LABORATORY   METHODS 

The  specimens  must  not  be  washed  out  in  running  water,  as 
it  removes  the  blood  on  which  the  color  depends.  An  excess  of 
blood  should  be  wiped  off  before  placing  the  specimens  in  No.  i . 
Specimens  lose  their  color  after  remaining  in  solution  for  a  few 
days,  but  the  color  soon  returns  after  the  specimens  are  trans- 
ferred to 

No.  2 

• 

Alcohol         . 4  parts 

Water I  part 

in  which  they  remain  for  3  to  5  hours,  after  which  they  are 
placed  in 

No.  3 

Alcohol,  95  per  cent, 
for  i  to  2  hours,  and  finally  are  permanently  preserved  in 

No.  4 

Potassium  acetate i  part 

Glycerin          ....        V       ....  2  parts 

Water  (distilled) 10  parts 

which,  before  use,  should  be  allowed  to  stand  for  48  hours  and 
then  filtered.  Keep  the  specimens  in  a  dark  place,  because  the 
light  fades  the  color  in  course  of  time.  (Texas  Med.  News., 
Vol.  X,  p.  10.) 

86.  Marlmann's  method  for  preserving  the  colors  of  anatom- 
ical specimens,  and  for  fixing  and  preserving  specimens  where 
it  is  desirable  to  avoid  alcohol. 

No.  i 

Sodium  fluorid          .......         5°  grammes 

Formaldehyd  (40  per  cent) 20  cc. 

Water io°°  cc. 

From  this  fluid  the  preparations  are  passed  into  the  following 
mixture  for  preservation  :  — 


USEFUL   FORMULA  AND  TABLES  293 

Glycerin  (28°  B) 500  cc. 

Water 1000  cc. 

Magnesium  chlorid 100  grammes 

Sodium  fluorid          .......  20  grammes 

Objects  for  sections  should  be  washed  three  or  four  times  in 
water,  and  then  treated  with  the  usual  grades  of  alcohol.  {Jour. 
App.  Mic.,  Vol.  IV,  p.  1582.) 

87.  Decalcifying  solution  :  — 

Nitric  acid 3  parts 

Chromic  acid 70  parts 

Water 200  parts 

After  bone  becomes  soft  enough  to  permit  a  needle  to  enter, 
wash  in  water  and  harden  in  alcohol. 

88.  Another:  — 

(1)  Imbed  in  celloidin  in  the  usual  way. 

(2)  Harden  by  slow  evaporation  in  alcohol  or  chloroform. 

(3)  The  celloidin  blocks  are  then  immersed  in  mixture  of 
alcohol  90  per  cent  and  nitric  acid ;  the  proportion  of  latter  is 
regulated  by  the  amount  of  calcareous  matter  in  specimen  (10  to 
100  of  alcohol). 

(4)  Renew  decalcifying  fluid  from  time  to  time. 

(5)  Remove  excess  of  acid  by  washing. 

(6)  Immerse  in  alcohol  90  per  cent,  changed  for  several 
days. 

(7)  Section  the  celloidin  block  after  removing  acid.     (Bull. 
Soc.  Beige  de  Mic.,  XXIII,  p.  159.) 

89.  Lacquering  microscope  tubes  and  stands  :   Cork  the  tube 
to  prevent  the  ingress  of  the  cleaning  solutions  and  then  wash 
in  the  following  soap  mixture  to  remove  old  lacquer :  — 

Shaved  castile  soap I  part 

Alcohol  95° 3  parts 

Benzol 3-4  parts 

Liquid  potassae I  part 

Polish  with  Putz  pomade  and  finish  with  chamois  skin  sprinkled 
with  finest  levigated  chalk.  Wash  a  second  time  in  soap  mix- 


294        BIOLOGICAL  LABORATORY  METHODS 

ture.  Place  then  in  hot  water,  wipe  dry,  and  apply  either  one 
of  the  following  lacquers  with  a  camel's-hair  brush,  with  the 
strokes  all  in  the  same  direction.  The  water  must  not  be  above 
66°  to  71°  C.,  otherwise  there  is  danger  of  blistering  the  lacquer. 

(1)  Pale  gold  lacquer  :  — 

Shellac  quite  clean    " I  part 

Ground  turmeric I  part 

Alcohol 4  parts 

Mix  and  set  aside  in  a  warm  place  with  frequent  agitation 
until  lacquer  is  dissolved.  Decant  clear  liquid  and  preserve 
for  use. 

(2)  Yellow  gold  :  — 

Shellac 25  parts 

Turmeric 4  parts 

Dragon's  blood     . I  part 

Alcohol  95° 70  parts 

Digest  for  eight  days  with  frequent  agitation ;  decant  and 
filter. 

(3)  Deep-red  gold  :  — 

Spanish  anatto 2  parts 

Turmeric  powdered       .......  30  parts 

Saunders's  red 3  parts 

Alcohol  95° 480  parts 

Infuse  in  the  cold  for  24  to  36  hours,  shaking  occasionally. 
Let  stand  until  settled  and  then  add :  — 

Shellac 60  parts 

Sanderac       .........  15  parts 

Mastic 15  parts 

Canada  balsam .  15  parts 

When  dissolved,  add  10  parts  of  oil  of  turpentine.  Upon 
highly  polished  brass  this  gives  a  deep-red  gold  color  that  is 
lasting  and  very  handsome.  (F.  L.  JAMES.) 


USEFUL   FORMULA  AND  TABLES 


295 


90.    Paste  for  mounting  botanical  specimens  : — 

Tragacanth  in  powder  ....... 

Gum  arabic 

Glycerin 

Water 

Bichlorid  of  mercury 

Boiling  water        ........ 


30  parts 

20  parts 

30  parts 

60  parts 

I  part 

240  parts 


Mix  the  gums  with  glycerin  and  water  in  a  mortar  with  vig- 
orous stirring.  Dissolve  the  bichlorid  of  mercury  in  boiling 
water  and  add  to  the  gum  mixture.  When  cold  put  in  a  few 
drops  of  oil  of  cloves.  (Nat.  Drug.,  p.  209,  1889.) 

91.  Cement  for  joining  parts  of  apparatus,  which  will  resist 
heat,  water,  acids,  and  oils. 

Mix  concentrated  glycerin  with  finely  powdered  litharge  to  a 
thick  paste.  Glass,  metal,  and  wood  can  be  cemented  together 
by  it.  {Jour.  Mic.  and  Nat.  Set.,  Vol.  II,  p.  53.) 

92.  Another:     Gum  elastic  dissolved  in  benzin  until  liquid 
has  syrupy  consistency.     White  lead  and  linseed  oil  varnish  are 
rubbed  up  to  a  paste  and  mixed  with  the  gum.     Cements  glass 
to  glass  or  wood  to  glass.     Good  for  aquarium.     (Roy.  Mic. 
Jour.) 

93.  Apparatus  for  removing  air  bubbles  from  slide  in  mount- 
ing microscopical  objects :  Consists  of  a  slip  of  plate  glass  (Fig. 


FlG.  124.  —  Apparatus  for  removing  Air  Bubbles  from  Slide. 

124)  measuring  4"  x  i^'r,  to  which  a  frame  of  wood  has  been  ce- 
mented. The  frame  is  of  such  a  size  as  to  allow  the  ordinary 
glass  micro  slips  being  placed  on  it.  In  one  side  of  the  frame 


296  BIOLOGICAL  LABORATORY   METHODS 

is  a  hole  bored,  and  one  end  of  a  piece  of  india-rubber  tube 
measuring  6  inches  long  by  T3g-  inch  in  diameter  is  cemented 
to  it.  A  piece  of  glass  tubing  B  i  inch  in  length  is  closed 
at  one  end ;  and  a  small  hole  is  bored  through  it  at  about  a 
quarter  of  an  inch  from  the  closed  end.  The  open  end  of  the 
glass  tube  is  then  slipped  into  the  free  end  of  the  rubber 
tube,  and  the  two  so  arranged  that  the  hole  in  the  former  shall 
be  just  covered  by  the  india-rubber.  To  use  the  apparatus, 
place  the  mount  containing  the  air  bubbles  in  the  wooden  cell 
and  cover  it  with  a  second  slip  of  glass,  the  edges  of  which 
have  been  previously  greased  with  tallow.  Exhaust  the  air  by 
drawing  through  the  tube.  The  valve  formed  by  the  hole  will 
prevent  the  reentrance  of  the  air,  and  any  bubbles  in  the  mount 
will  quickly  disappear.  (J.  H.  COOKE,  in  Jour.  App.  Mic.,  Vol. 
II,  p.  621.) 

94.  Cutting  glass  tubes  and  bottles:    Cut  strips  of  blotting- 
paper   from   \  to  \  inch    in  width,  wet  and  wrap  around  the 
bottle  \  to  f  inch  apart.     Throw  a  fine  flame  2  or  3  inches  long 
between  the  wet  papers,  revolving  the  bottle  slowly.     Within  a 
minute  the  glass  will  break  with  a  clear  cut  along  the  line  fol- 
lowed by  the  flame.     (PROFESSOR  WILLIAM  THOMPSON,  Jour. 
Mic.  and  Nat.  Sd.) 

95.  Paste  for  labels  :  — 

Gum  arable 15.0    parts 

Tragacanth 7.5    parts 

Glycerin 45.0    parts 

Thymol 0.3    part 

Alcohol 3.75  parts 

Water  to  make  up  to 120.0    parts 

Dissolve  gum  arabic  in  15  parts  of  water,  rub  up  tragacanth 
in  30  parts  of  water.  Then  mix  and  strain.  Add  the  glycerin 
and  finally  the  alcohol  and  thymol.  (Zeit.  f.  Angew.  Mikr., 

in.) 

96.  Loosen  stoppers  by  placing  a"  little  glycerin  around  the 
stopper  •  and  smearing  some  glycerin  on  the  portion  of  stop- 


USEFUL   FORMULA  AND   TABLES 


297 


per    going    into    the    bottle   will    prevent    it    from    becoming 
tightened. 

97.  Refractive  indexes  and  alcohol  solvent  powers  of  clear- 
ing and  mounting  media.  (E.  M.  BRACE,  in  Jour.  App.  Mic., 
Vol.  I,  p.  220.) 


Substances 

Refractive  Index 

Per  cent  of  Alcohol  which 
Substance  will  dissolve 

Cloroform       .... 

459 

80% 

Oil  of  Cedar  .... 

.466 

95% 

Turpentine     .... 

.469 

100% 

Oil  of  Orange 

.472 

95% 

Oil  of  Origanum     . 

479 

95% 

Xylene  

493 

95% 

Benzin    

495 

95'% 

Oil  of  Thyme 

.496 

95% 

Oil  of  Cedarwood  . 

.502 

95% 

Xylene  Balsam 

•524 

— 

Oil  of  Cloves  .... 

•532 

80% 

Oil  of  Anilin  .... 

.585 

60% 

Oil  of  Cassia  .... 

.602 

80% 

98.  Retouching  formula  for  negatives  :  Equal  parts  of  gold 
and  benzol  make  an  excellent  cold  varnish  for  negatives,  which 
gives  a  surface  ready  for  the  retouching  pencil  without  further 
preparation.     (Wilson's  Photo.  Mag.,  December,  1901.) 

99.  To  title  negatives  :  — 


A.    Sugar 
Glycerin 
Water 


I  part 

3  parts 

10  parts 


B.    Mercuric  chlorid         .         .         .         .         .         .         .       i  part 

Mercuric  nitrate          .......       2  parts 

Alcohol .         .     10  parts 

Mix  equal  parts  of  A  and  B  and  write  on  paper.  Transfer  the 
writing  to  the 'film  by  pressing  it  into  close  contact.  (Wilson's 
Photo.  Mag.,  Vol.  III.) 


298        BIOLOGICAL  LABORATORY  METHODS 

100.    A  black  finish  for  table-tops  :  — 

No.  I.   Copper  sulphate  .         .         .  125  grammes 

Potassium  chlorate 125  grammes 

Water          .         .         .         .         .         .         .     1000  grammes 

Boil  until  dissolved. 

No.  2.    Anilin  hydrochlorate 150  grammes 

Water 1000  grammes 

or 

Anilin  oil 1 20  grammes 

Hydrochloric  acid 180  grammes 

Water 1000  grammes 

Apply  to  the  table-top  two  coats  of  No.  i  while  hot,  and  as 
soon  as  this  first  coat  is  dry  apply  a  second  coat.  Then  put  on 
two  coats  of  No.  2  and  allow  to  dry.  Rub  in  with  a  cloth  a  thin 
coating  of  linseed  oil ;  rapid  and  lengthy  rubbing  in  of  the  oil 
will  give  the  polish  to  the  wood.  Wash  out  the  surplus  chemi- 
cals with  hot  soap-suds.  The  finish  left  is  a  deep  ebony  black. 
It  is  claimed  that  acids  will  not  stain  this  table  finish  and  that 
anilin  stains  can  be  washed  off  by  alcohol.  (Bot.  Zeit.,  54,  326, 
and  Bot.  Gaz.,  24,  66.) 

i  o  i .    Another  table-top  finish :  — 

No.  I.    Sodium  chlorate 67  grammes 

Copper  chlorate 67  grammes 

Water I  liter 

No.  2.   Anilin  chlorate '5°  grammes 

Water i  liter 

Paint  the  table  with  No.  i  and  follow  with  No.  2  and  allow  to 
dry.  Three  applications  will  give  a  dense  black.  After  the 
surface  is  dry,  it  can  be  finished  with  oil.  (J.  R.  SLONAKER.) 


USEFUL   FORMULA  AND   TABLES 


299 


102.  —  CONVERSION  OF  BRITISH  AND  METRIC  MEASURES 

Computed  by  Mr.  E.  M.  Nelson  from  the  New  Coefficient  obtained  by  order 
of  the  Board  of  Trade  in  1896.     {Jour.  Roy.  Mic.  Soc.} 

LINEAL 


METRIC  INTO  BRITISH 

BRITISH  INTO  METRIC 

ft.    in. 

mm.    in. 

mm.    in. 

in. 

mm. 

in. 

mm. 

I  .OOOO39 

I   -03937° 

41  1.614175 

I 

25-399978 

A 

1  7.779985 

2  .000079 

3  .000118 
4  .000157 
5  .000197 
6  .000236 

2   .078740 

3  .118110 
4  .157480 
5  .196851 
6  .236221 

42  1.653545 

43  1.692915 
44  1.732285 
45  1-771655 
46  1.811025 

2 

3 
4 
5 

50.799956 
76.199934 
IOI.5999I2 
126.999890 

A 

TV 
1\ 

A 

22.859980 
2.309089 
2.116665 
10.583324 

7  .000276 

7  -275591 

47  1-850395 

6 

152.399868 

A 

14.816654 

8  .000315 

8  .314961 

48  1.889765 

7 

177.799846 

ii 

23.283313 

9  .000354 
10  .000394 

ii  .000433 

12  .000472 
13  .000512 

9  -354331 
10  -393701 
ii  .433071 

12   .472441 
13   .5Il8lI 

49  1.929136 
50  1.968506 

51  2.007876 
52  2.047246 
53  2.086616 

8 
9 

10 

ii 

203.199824 
228.599802 
253.999780 
279.399758 

TV 

& 
1 
1-5 

TV 

L953844 
1.814284 

1.693332 
1.587499 

14  .000551 
15  .000591 

14   .551182 
15   .590552 

54  2.125986 
55  2.165356 

i  ft. 

304-799736 

tV 

A 

4.762496 
7-937493 

1  6  .000630 

16  .629922 

56  2.204726 

i  yd. 

914.399208 

7 

II.II249O 

17  .000669 

17  .669292 

57  2.244096 

To 
9 

14.  2874.87 

1  8  .000709 
19  .000748 

1  8  .708662 
19  .748032 

58  2.283467 
59  2.322837 

in. 
i 

mm. 
12.699989 

T^ 
tt 

•i^f-.^<->/  <-f  °/ 
17.462485 

20  .000787 

20  .787402 

60  2.362207 

| 

8.466659 

it 

20.637482 

21  .000827 

21   .826772 

61  2.401577 

3 

I 

T^    j  :/ 

l6-9333I9 

u 

23.812479 

22  .OOO866 

22   .866142 

62  2.440947 

i 

6.349994 

TV 

I.494II6 

23  .OOO9O6 
24  .000945 
25  .000984 

23  -9055  1  3 

24   .944883 
25   .984253 

63  2.480317 
64  2.519687 
65  2.559057 

4 

1 
I 

19.049983 
5.079996 

TV 

A 

i 

I.4IIIIO 

1.336841 
I  26QOQQ 

26  .OOIO24 

26  1.023623 

66  2.598427 

I 

10.159991 

^V 

1  •*vry:7SrJf 

27  .OOIO63 

27  1.062993 

67  2.637798 

| 

15.239987 

?T 

1.209523 

28  .OOIIO2 

28  1.102363 

68  2.677168 

| 

20.319982 

A 

LI54544 

29  .OOII42 

29  I.I4I733 

69  2.716538 

i 

i 

1.104347 

30  .OOIlSl 

3O  I.l8lIO3 

70  2.755908 

£ 
| 

4.233330 
21.166648 

2  3 

A 

1.058332 

31  .001220 
32  .OOI26O 

33  .001299 

31  1.220473 
32  1.259844 

33  1.299214 

71  2.795278 
72  2.834648 
73  2.874018 

D 

\ 

\ 

3.628568 
3-  i  74997 

A 

A 

i 

1.015999 
.846666 

34  .001339 

34  1-338584 

74  2.913388 

t 

9.524992 

A 

•725714 

35  .001378 

35  1-377954 

75  2.952758 

1 

15,874986 

A 

•634999 

36  .001417 
37  .001457 
38  .001496 
39  -001535 

36  1.417324 
37  1-456694 
38  1.496064 

39  1-535434 

76  2.992129 

77  3.03  H99 
78  3.070869 
79  3-110239 

* 
\ 
TV 

22.224980 
2.822220 
2.539998 

A 
A 
A 

•564444 
.508000 
.461818 

40  .001575 

40  1.574805 

80  3.149609 

A 

7.619993 

i 

^r<y 

•423333 

300 


BIOLOGICAL   LABORATORY   METHODS 


CONVERSION  OF  BRITISH  AND  METRIC  MEASURES   CONTINUED 

LINEAL 


METRIC  INTO  BRITISH 

BRITISH  INTO  METRIC 

ju.     in. 

mm.    in. 

mm.     in. 

in.      mm. 

in. 

mm. 

41  .OOl6l4 
42  .001654 

43  .001693 
44  .001732 

81  3.188979 
82  3-228349 
83  3-267719 
84  3.307089 

96  3-779531 

g7  3.818901 
g8  3.858271 
99  3.897641 

Js  .390769 

TS   .362857 

7V  .338666 

til 

III 

.033867 
.031750 
.029882 

45  .001772 

85  3.346460 

•fa   .317500 

uw 

.028222 

46  .001811 

86  3-385830 

dm.     in. 

fa   .298823 

,b 

.026737 

47  .001850 
48  .001890 
4g  .001929 
50  .001969 

60  .002362 
70  .002756 
80  .003150 

87  3.425200 
88  3.464570 

8g  3-503940 

90  3.543310 

gi  3.582680 
g2  3.622050 
g3  3.661420 

i  3-937OII3 

2   7.8740226 

3  11.8110339 
4  15.7480452 
5  19.6850565 
6  23.6220678 
7  27.5590791 

•fa   .282222 
•fa   .267368 
Tfo   .254000 

lis  .169333 
^J7  .127000 
•sfaf  .101600 

in. 

25.399978 
12.699989 
8.466659 
6-349994 

go  .003543 

94  3.70079I 

8  31.4960904 

•dv  .084667 

-5V5T) 

5.079996 

100  .003937 

95  3.740161 

9  35«433Ioi7 

?h  -072571 

^7(J(J 

4-233330 

200  .007874 
3OO  .OIlSlI 

itro  .063500 

Tinnr 

3.628568 

4OO  .015748 

¥^  .056444 

loViy 

3-  1  7499  7 

500  .019685 

•sbv  .050800 

Wfrv 

2.822220 

6OO  .023622 

-SI-Q     .046182 

T7777T5- 

2.539998 

700  .027559 
800  .031496 

^A(5   .042333 

T5TT(TO' 

I.693332 

goo  .035433 

i  meter  =  3.2808428  ft. 

SS<5   ^39077 

YOWG 

1.269999 

iooo=(imm.) 

=  1.09361426  yd. 

Tlhr  .036286 

msfam 

1.015999 

103. — METRIC  SYSTEM  OF  WEIGHTS  AND  MEASURES 

MEASURES    OF    LENGTH 


DENOMINATIONS  AND  VALUES 


EQUIVALENTS  IN  USE 


Myriameter  .  .  . 
Kilometer  .  .  .  . 
Hectometer  .  .  . 
Dekameter  .  .  . 
Meter  .  . 

10,000  meters 
1,000  meters 
100  meters 
•         10  meters 
i  meter 

6.2137    miles 
.62137  mile>  or  3>28o  ft.  10  in. 
328.             feet  and  I  inch 
393.7          inches 
•JQ  ^7        inches 

Decimeter  .... 
Centimeter  .  .  . 
Millimeter  .... 

i-ioth  of  a  meter 
i-iooth  of  a  meter 
i-ioooth  of  a  meter 

3.937      inches 

•3937    inch 
.0394    inch 

USEFUL   FORMULA  AND   TABLES 


301 


MEASURES    OF    SURFACE 


DENOMINATIONS  t 

L.ND  VALUES 

EQUIVALENTS  IN  USE 

Hectare         

10,000  square  meters 

2.471  acres 

Are    

100  square  meters 

119.6      square  yards 

Centare                       .      .  .  . 

i  square  meter 

1,550.        square  inches 

MEASURES    OF    VOLUME 


DENOMINATIONS  AND  VALUES 


NAMES 

No.  OF 
LITERS 

CUBIC 
MEASURES 

DRY  MEASURE 

WINE  MEASURE 

Kiloliter  or  stere    . 
Hectoliter    

I,OOO 
IOO 

I  cm. 
i-ioth  cm. 

1.308     cubic  yards 
2.          bu.  and  3.35 

264.17      gallons 

Dekaliter            .  .  . 

IO 

10  cd. 

pecks 
9.08      quarts 

26.417    gallons 
2  6417  gallons 

Liter    

I  cd. 

.908    quart 

1.0567  quarts 

Deciliter 

I  —  IO 

i—  loth  cd 

6*1023  cubic  inches 

845    gill 

Centiliter     .      . 

I—  IOO 

IO  CC. 

.6102  cubic  inch 

338    fluid  oz 

Milliliter  

I—  IOOO 

I   CC. 

.061     cubic  inch 

.27      fl.  drm. 

EQUIVALENTS  IN  USE 


WEIGHTS 


DENOMINATIONS  AND  VALUES 


EQUIVALENTS 
IN  USE 


NAMES 

NUMBER  OF 
GRAMS 

WEIGHT  OF  VOLUME  OF 
WATER  AT  ITS  MAXI- 
MUM DENSITY 

AVOIRDUPOIS 
WEIGHT 

Millier  or  Tonneau  .  .  . 
Quintal    
Myriagram    

I  ,OOO,OOO 
IOO,OOO 
IO,OOO 

I  cm. 
I  hi. 
10  1. 

2204.6         pounds 
220.46      pounds 
22.046    pounds 

Kilogram  or  Kilo  .... 
Hectogram    

I,OOO 
IOO 

I  1. 
I  dl. 

2.2046  pounds 
3C274.  ounces 

Dekagram  

IO 

IO  CC. 

.3527  ounce 

Gram 

I 

I   CC 

I  £  4.^2    grains 

Decigram          

I-IO 

i—  loth  of  a  cc 

I.C432  grains 

Centigram  

I—  IOO 

10  cmm. 

.154^  grain 

Milligram    ...         . 

I—  IOOO 

I  cmm 

oi  ^4.  grain 

302 


BIOLOGICAL   LABORATORY   METHODS 


104.  —  UNITED  STATES  WEIGHTS  AND  MEASURES 


12  inches  =  I  foot. 

3  feet  =  I  yard. 
5.5  yards  =  I  rod. 
40  rods  =  I  furlong. 

8  furlongs  =  I  mile. 


LINEAL    . 

Inches  Feet  Yards             Rods         Fur. 

12   =  I 

36  -  3     =  i 

198  =  16.5  =  5.5   =       i 

7,920  =  660     =  220      =     40    =     i 

63,360  =  5,280     =  1,760      =  320    —    8 


Mile 


144  sq.  in.  =  I  sq.  ft. 
9  sq.  ft.  =  i  sq.  yard. 
30.25  sq.  yd.  =  I  sq.  rod. 
40  sq.  rods  =  I  sq.  rood. 
4  sq.  roods  =  I  acre. 
640  acres  =  I  sq.  mile. 


SURFACE LAND 

Feet  Yards                Rods           Roods    Acres 

9=  I 

272.25  =  30.25  =       I 

10,890  =  I,2IO  =      40  =     I 

43,560=         4*849=          l6o=         4=      I 
27,878,400  =  3,097,600  =  102,400  =  2,560  =  640 


VOLUME LIQUID 


4  gills  —  i  pint. 
2  pints  =  I  quart. 
4  quarts  =  I  gallon. 


Gills          Pints        Gallon         Cubic  Inches 

32     =    8    =       i       =       231 


Gallon 


Pints. 


Ounces 

=         128 

16        = 


FLUID 
Drachms 


Minims          Cubic  Centimeters 


I,O24  —  61,440  = 

128  =  7,680  = 

8  -=  480  = 

I  =  60  = 


1 6  ounces,  or  a  pint,  is  sometimes  called  a  fluid  pound. 


473.179 
29-574 


TROY    WEIGHT 


Pound 

Ounces 

Pennyweights 

Grains 

Grams 

I             = 

12 

240           = 

5,760 

=       373-24 

I 

=                20            = 

480 

=          3LIO 

I            = 

24 

I.56 

USEFUL   FORMULA  AND  TABLES  303 

APOTHECARIES'   WEIGHT 


ft 

3 

3 

3 

gr- 

Pound 

Ounces 

Drachms 

Scruples 

Grains 

Grams 

I            — 

12 

=         96         = 

288        = 

5,760      = 

373.24 

I 

=          8        = 

24        = 

480      = 

3I.IO 

i         = 

3      = 

60      = 

3-89 

i      = 

20       = 

1.30 

I       = 

.07 

The  pound,  ounce,  and  grain  are  the  same  as  in  Troy  weight. 
All  chemicals  are  usually  sold  by 


AVOIRDUPOIS    WEIGHT 

Pound                Ounces                Drachms                Grains  (Troy)  Grams 

i        =         1 6        =        256         =         7,000          =  453.6o 

i                     1 6         =            437-5       =  28.35 

i         =              27.34     =  1.77 

i           =  .07 


UNITED    STATES    FLUID    MEASURE 
Gal.      Pints     Ounces      Drachms        Minims  Cu.  In.  Grains  Cc. 

i    =    8  =   128  =   1,024  —  61,440  =  231.          =  58,328.886    =3,785.44 

i    —     16  =      128  =  7,680  =  28.875    =  7,291.1107  =    473.18 

i   =          8  =  480  =  i  8347  =  455.6944  =      29.57 

I  =  60  =  0.2256  =  56.9618  =       3.70 


IMPERIAL    BRITISH    FLUID    MEASURE 
Gal.      Pints     Ounces      Drachms        Minims  Cu.  In.  Grains  Cc. 

i  ==  8  =  160  =  1,280  =  76,800  =  277.27384  =  70,000   =  4,543.732 

i  =  20  =   160  =  9,600  =  34.65923  =  8,750   =  567.966 

i  =    8  =  480  =  1.73296  =   437-5  =   28.398 

i  =  60  =  0.21662  =    54.69  =    3*55° 


304 


BIOLOGICAL  LABORATORY   METHODS 


105.  —  TABLE  SHOWING  CORRESPONDING  DEGREES  ON  THE 
SCALE  OF  THE  FAHRENHEIT  AND  CENTIGRADE  THERMOMETERS 


FAHR. 

CENT. 

FAHR. 

CENT. 

FAHR. 

CENT. 

FAHR. 

CENT. 

FAHR. 

CENT. 

FAHR. 

CENT. 

32. 

o. 

62. 

16.6 

91.4 

33- 

121. 

49-4 

152. 

66.6 

182. 

83.3 

33- 

-5 

62.6 

17. 

92, 

33-3 

122. 

152.6 

67. 

183. 

83.8 

33-8 

63. 

17.2 

93- 

33-8 

I23. 

5°-5 

153. 

67.2 

183.2 

84. 

34- 

i.i 

64. 

93-2 

34- 

123.8 

154- 

67.7 

184. 

84.4 

35- 

1.6 

64.4 

18. 

94- 

34-4 

124. 

Si-1 

154-4 

68. 

I85. 

85- 

35-6 

2. 

65. 

18.3 

95- 

35- 

125. 

51.6 

155. 

68.3 

1  86. 

85.5 

36. 

2.2 

66. 

18.8 

96. 

35-5 

125.6 

52- 

156. 

68.8 

186.8 

86. 

37- 

2.7 

66.2 

19. 

96.8 

36. 

126. 

52.2 

156.2 

69. 

187. 

86.1 

37-4 

3- 

67. 

19.4 

97- 

36.1 

127. 

52-7 

157. 

69.4 

1  88. 

86.6 

38. 

3-3 

68. 

20. 

98. 

36.6 

127.4 

53- 

I58. 

70. 

188.6 

87. 

39- 

3-8 

69. 

20.5 

98.6 

37- 

128. 

53-3 

159- 

70-5 

189. 

87.2 

39-2 

4- 

69.8 

21. 

99. 

37-2 

129. 

53-8 

159.8 

71- 

190. 

87.7 

40. 

4.4 

70. 

21.  1 

100. 

37-7 

129.2 

54- 

I  60. 

71.1 

190.4 

88. 

41. 

5- 

21.6. 

100.4 

38. 

I30. 

54-4 

161. 

71.6 

191. 

88.3 

42. 

5-5 

ll'.e 

22. 

IOI. 

38.3 

55- 

161.6 

72. 

192. 

88.8 

42.8 

6. 

72. 

22.2 

102. 

38.8 

I32. 

55-5 

162. 

72.2 

192.2 

89. 

43- 

6.1 

73- 

22.7 

IO2.2 

39- 

132.8 

56. 

163. 

72.7 

193. 

89.4 

44- 

6.6 

73-4 

23- 

I03. 

39-4 

133. 

56.1 

163.4 

73- 

194. 

90. 

44.6 

7- 

74- 

23-3 

104. 

40. 

134- 

56.6 

164. 

73-3 

195- 

90-5 

45- 

7.2 

75- 

23.8 

105. 

40-5 

134.6 

57- 

165. 

73-8 

195-8 

91. 

46. 
46.4 

r 

75-2 
76. 

24. 
24.4 

105.8 
1  06. 

41. 
41.1 

135- 
I36. 

57-2 
57-7 

165.2 
1  66. 

74- 
74-4 

196. 
197. 

91.1 
91.6 

47- 

8.3 

77- 

25- 

107. 

41.6 

136.4 

58. 

167. 

75- 

197.6 

92. 

48. 

8.8 

78. 

25-5 

107.6 

42. 

58.3 

1  68. 

75-5 

198. 

92.2 

48.2 

9- 

78.8 

26. 

108. 

42.2 

^38.' 

58.8 

168.8 

76. 

199. 

92.7 

49- 

9-4 

79- 

.26.1 

109. 

42.7 

138.2 

59- 

169. 

76.1 

199.4 

93- 

10. 

80. 

26.6 

109.4 

43- 

I39. 

59-4 

170. 

76.6 

200. 

93-3 

51. 

10.5 

80.6 

27. 

1  10. 

43-3 

140. 

60. 

170.6 

77- 

201. 

93-8 

51.8 

n. 

81. 

27.2 

in. 

43-8 

141. 

60.5 

171. 

77-2 

2OI.2 

94. 

n.  i 

82. 

27.7 

III.  2 

44- 

I4I.8 

61. 

172. 

77-7 

2O2. 

94-4 

53- 

11.  6 

82.4 

28. 

112. 

44-4 

142. 

61.1 

172.4 

78. 

203. 

95- 

53-6 

12. 

83- 

28.3 

113- 

45- 

143- 

61.6 

78-3 

204. 

95-5 

54- 

12.2 

84. 

28.8 

114. 

45-5 

143.6 

62. 

174. 

78.8 

204.8 

96. 

55- 

I2.7 

84.2 

29. 

II4.8 

46. 

144. 

62.2 

174.2 

79- 

205. 

96.1 

55-4 

85. 

29.4 

"5- 

46.1 

H5- 

62.7 

175- 

79-4 

2O6. 

96.6 

56. 

57- 

13-3 
I3.8 

86. 
87. 

30- 
30-5 

116. 
116.6 

46.6 

47- 

145-4 
146. 

63- 

176. 

177. 

80. 
80.5 

2O6.6 
207. 

97- 
97-2 

57-2 

14. 

87.8 

117. 

47-2 

147. 

63^8 

177-8 

81. 

208. 

97-7 

58. 

14.4 

88. 

3I-1 

118. 

47-7 

147.2 

64. 

178. 

81.1 

208.4 

98. 

59- 

15- 

89. 

31-6 

118.4 

48. 

I48. 

64.4 

179. 

81.6 

209. 

98.3 

60. 

15-5 

89.6 

32. 

119. 

48.3 

I49. 

65. 

179.6 

82. 

2IO. 

98.8 

60.8 

1  6. 

90. 

32.2 

1  20. 

48.8 

150. 

65-5 

1  80. 

82.2 

2IO.2 

99- 

61. 

16.1 

91. 

32-7 

120.2 

49- 

150.8 

66. 

181. 

82.7 

211. 

99.4 

I5I. 

66.1 

181.4 

83- 

212. 

100. 

APPENDIX 

THE  ARRANGEMENT  OF  THE  LABORATORY  AND  ITS 
FURNITURE 

THE  proper  location  of  the  room  used  for  the  microscope  is  a 
matter  strongly  insisted  upon  as  important  by  most  microsco- 
pists,  some  contending  that  a  northern  exposure  is  absolutely 
necessary  to  insure  the  proper  adjustment  of  the  light,  while 
others  assert  that  a  western  or  southern  exposure  will  do  just  as 
well  if  the  windows  are  covered  with  white  shades  so  that  the 
light  will  be  uniformly  distributed  through  the  room  and  the 
glare  overcome.  The  latitude  in  which  the  laboratory  is  situated 
will  have  much  to  do  with  determining  this  important  question, 
but  there  is  no  doubt  of  the  fact  that  a  northern  light  produces 
much  more  pleasing  effects  upon  the  eye  of  the  observer,  since 
the  light  is  distributed  from  the  sky  and  is  softer  in  its  results. 

If  the  laboratory  is  also  to  be  used  for  purposes  demanding 
the  direct  rays  of  the  sun,  then  a  southern  exposure  will  be 
demanded  ;  in  this  case  the  even  distribution  of  the  light  can  be 
controlled  by  means  of  white  shades  on  the  windows,  adjusted 
to  rollers. 

The  laboratory  should  be  well  lighted  with  large  windows  so 
situated  as  to  supply  an  ample  amount  of  illumination  at  all 
hours  of  the  day.  The  ventilation  must  be  looked  after  and  put 
in  the  best  possible  condition,  so  that  all  deleterious  gases  from 
the  lungs  of  the  students  and  from  the  chemical  combinations 
which  may  be  produced  in  the  laboratory  may  be  carried  off  as 
rapidly  as  possible.  It  is  important  to  have  the  heating  appara- 
tus so  adjusted  that  the  temperature  will  be  normal  at  all  times 
of  the  year ;  in  the  winter  by  the  regulation  of  registers,  and  in 
the  summer  by  electric  fans  or  other  like  means.  The  strictest 
x  305 


306  APPENDIX 

attention  must  be  paid  to  the  entrance  of  dust  particles  into  the 
laboratory,  as  nothing  so  greatly  injures  apparatus  and  delicate 
experiments  as  this  enemy  of  scientific  research.  Provision 
must  be  made  in  the  laboratory  for  illuminating  and  heating 
gas,  and  it  will  be  convenient  to  have  gas  fixtures  on  each  table 
in  easy  reach  of  the  student  for  many  purposes  during  the 
preparation  of  the  specimen  for  mounting.  Electric  lamps 
with  ground-glass  globes  mounted  on  movable  bases  will  be 
convenient  during  dark,  cloudy  days ;  or,  if  electricity  is  not 
available,  Welsbach  mantles  will  give  good  light  with  suitable 
gas-burners. 

The  room  should  contain  an  ample  supply  of  water,  with  a 
properly  constructed  sink  located  near  the  centre  of  the  labora- 
tory, or  in  easy  reach  of  all  members  of  the  class.  This  sink 
should  have  several  faucets,  and  it  should  be  lined  with  lead  to 
withstand  the  action  of  chemicals.  The  laboratory  must  have 
shelving  for  storing  the  material  and  apparatus  not  in  immediate 
use  by  the  students.  Individual  lockers  are  necessary,  either 
attached  to  the  tables  or  grouped  together  in  one  portion  of  the 
room  where  the  reagents  and  apparatus  of  the  workers  may  be 
safely  kept  free  from  dust  and  interference,  and  in  order  that 
each  student  may  be  rigidly  held  responsible  for  the  outfit. 
The  laboratory  locker  in  use  in  Cornell  University  and  described 
in  the  Journal  of  Applied  Microscopy^  will  be  found  very  efficient 
in  this  connection.  The  description  given  of  these  lockers  is  as 
follows :  "  The  lockers,  like  all  of  the  woodwork  in  the  labora- 
tory, are  of  quartered  oak.  Each  is  provided  with  a  combination 
lock,  the  combination  of  which  is  known  to  the  student  to  whom 
it  is  assigned  and  the  laboratory  director  only.  The  lockers 
themselves  contain  no  fixtures  whatever,  being  provided  with 
six  pairs  of  slides.  The  laboratory  owns  a  large  number  of 
reagent  boards  and  drawers,  all  of  which  are  exactly  the  same 
size  and  of  the  proper  width  to  fit  into  the  lockers.  Each 
student  is  provided  with  as  many  of  these  reagent  boards  or 
drawers  as  his  work  requires,  and  all  reagent  bottles,  glassware, 

l  Jour.  App.  Mic.,  Vol.  I,  p.  26. 


APPENDIX  307 

accessory  apparatus,  material,  etc.,  are  always  to  be  kept  on 
these  boards  or  in  the  drawers.  The  reagent  boards  are  of 
light  pine  one  and  seven-eighths  inches  thick,  and  have  on  one 
side  depressions  in  which  the  bottles  or  dishes  containing 
reagents,  specimens,  etc.,  are  set." 

The  Table.  —  This  necessary  part  of  the  furnishing  of  a  labora- 
tory has  assumed  almost  as  many  shapes  and  styles  as  there 
are  laboratories  in  the  country.  The  form,  height,  and  dimen- 
sions are  subjects  greatly  discussed  by  microscopists ;  but  the 
general  consensus  of  opinion,  however,  gives  the  following  rules 
in  regard  to  the  laboratory  table  :  — 

1.  The  student  should  sit  rather  than  stand  at  the  table. 

2.  Stools  rather  than  chairs  are  preferable,  because  of  the 
reduced  space  they  occupy,  and  the  ease  with  which  the  student 
can  move  to  and  from  the  table.     Stools  with  revolving  seats 
adjustable  for  height  are  best. 

3.  When  drawers  are  used  in  the  tables,  combination  locks  are 
preferred  for  many  reasons  that  are  obvious  to  the  thoughtful 
laboratory  director  who  has  had  experience  in  the  use  of  keys 
which  are  half  the  time  in  a  "  lost  "  condition. 

4.  The  tops  may  be  either  of  wood,  slate,  or  glass,  to  suit  the 
whim  of  the  instructor.     The  advantages  and  disadvantages  of 
each  will  be  given  farther  on. 

5.  If  the  space  in  the  laboratory  will  permit,  it  is  best  to  give 
each  student  a  table  to  himself.      In  small   laboratories  this 
would  not  be  feasible.     Care  should  be  taken,  however,  not  to 
crowd  the  manipulators. 

6.  Each  student  should  have  a  drawer  in  the  table  for  his 
own  use,  or  a  locker  in  the  near  laboratory  within  convenient 
reach,  in  which  he  may  secure  the  delicate  dissecting  instru- 
ments furnished  him  by  the  director. 

The  experience  of  the  American  microscopists  has  developed 
a  variety  of  tables,  which  may  be  classified  under  the  three  fol- 
lowing groups : — 

i.  Simple  small  tables  suitable  for  two  undergraduate  stu- 
dents. The  tops  to  be  20  x  54  inches  in  dimension. 


308 


APPENDIX 


2.  The  table  shown  in  Figs.  125  and  126  is  constructed  for  two 
students.    It  contains  drawers  at  A,  B,  F,  and  G ;  compartments 
at  C,  D  for  the  microscopes  when  not  in  use  ;  shelves  at  J5,  E\ 
with  doors  opening  at  each  end  of  the  table  at  H. 

3.  Figure  127  shows  a  table  popular  in  many  laboratories  in 
this  country.     The  lower  portion  of  the  figure  represents  a  top 
view  of  the  table,  and  the  upper  part  a  side  view.     The  dimen- 
sions are  given  in  the  illustration.     This  table  is  constructed  to 


-54-" 


-20 -> 


( — \ 
F 


FIG.  125.  —  Laboratory  Table,  Side  View. 


FIG.  126.  —  End  View. 


accommodate  seven  students,  and  the  shape  will  permit  the 
light  from  the  window  to  reach  each  microscope  without  inter- 
ference by  the  neighboring  students. 

The  material  used  in  the  construction  of  the  tops  of  the  labo- 
ratory tables  is  also  a  matter  of  considerable  controversy  among 
microscopists.  Wood,  slate,  and  glass  have  each  earnest  and 
strong  advocates.  If  wood  is  used,  the  top  must  be  prepared  to 
prevent  its  disfigurement  by  stains  and  other  chemicals  which 
will  soon  mar  its  surface  and  render  it  unsightly  and  unpleasant 
to  the  instructor,  whose  motto  should  be,  a  clean  laboratory  in 
all  of  its  approaches  and  appurtenances.  The  following  methods 
of  procedure  are  recommended  to  give  impervious  surfaces  to 


APPENDIX 


309 


•o 


-5-8- 


--30- 


FIG.  127.  —  Laboratory  Table,  Top  and  Side  Views. 


3IO  APPENDIX 

the  action  of  acids  and  other  chemicals  generally  used  in  the 
laboratory:  — 

i.  This  method  will  produce  a  black  stain  and  finish  which 
for  many  purposes  is  desirable  in  biological  work.  To  secure 
this  result  make  two  solutions,  the  following :  — 

No.  i 

Copper  sulphate     .         .         .         .         .         .         .125  grammes 

Potassium  chlorate 125  grammes 

Water    .  1000  grammes 

Boil  until  the1  salts  are  dissolved. 

No.  2 

Anilin  hydrochlorate 150  grammes 

Water 1000  grammes 

Or  — 

Anilin  oil 120  grammes 

Hydrochloric  acid 180  grammes 

Water 1000  grammes 

Apply  two  coats  of  solution  No.  i  while  hot,  and  when  dry 
spread  on  two  coats  of  No.  2,  and  after  the  wood  is  thoroughly 
dried  rub  on  raw  linseed  oil.  A  continued  rubbing  of  the  oil 
will  yield  a  polish,  and  the  deep-black  color  is  produced  by  wash- 
ing out  the  extra  chemicals  with  hot  soap-suds.  It  is  claimed  for 
this  method  that  the  wood  will  resist  the  action  of  concentrated 
chemicals.1 

Another  method  is  recommended  which  will  accomplish  prac- 
tically the  same  results.  This  has  been  suggested  by  Professor 
Dodge  of  Rochester  University,  and  consists  in  making  a  liberal 
application  to  the  freshly  planed  tops  of  a  solution  made  by 
boiling  logwood  chips  in  an  iron  kettle.  This  solution  must  be 
concentrated.  After  the  first  coat  dries  the  second  is  applied, 
which  is  also  followed  by  a  strong  solution  of  sulphate  of  iron 
in  hot  water.  When  dry  the  table  is  thoroughly  sand-papered, 
and  hot  paraffin  of  high  melting-point  (55°  to  60°  C.)  is  poured 

i  Jour.  Atf>.  Mic.,  Vol.  I,  p.  145. 


APPENDIX  311 

on.  The  paraffin  is  well  rubbed  into  the  wood  by  means  of  a 
hot  flat-iron.  When  the  surface  of  the  table  becomes  cool,  the 
superfluous  paraffin  is  scraped  off  by  a  thin  piece  of  steel  with  a 
straight  edge. 

Professor  F.  R.  Wright  of  Cornell  University  prefers  the  use 
of  glass  tops  for  tables,  and  in  describing  their  use  in  his  labora- 
tory he  makes  the  following  comments :  "  The  table  is  covered 
with  corrugated  rubber  matting,  which  is  of  the  same  size  as  the 
top  of  the  table,  and  on  this  pad  is  placed  a  piece  of  plate  glass 
which  projects  about  half  an  inch  beyond  the  edge  of  the  table. 
The  rubber  matting  prevents  the  glass  slipping,  insures  to  a  cer- 
tain extent  against  breakage,  offers  a  good  background,  and 
takes  up  much  of  the  vibration.  The  glass  removes  the  diffi- 
culties met  with  in  the  use  of  either  wood  or  slate ;  it  can  be 
thoroughly  disinfected,  is  easily  cleaned,  and  is  not  unpleasant 
for  one  at  work."1 

The  advocates  of  the  use  of  slate-top  tables  claim  all  the 
advantages  for  them  that  are  admitted  as  belonging  to  glass, 
with  the  additional  advantage  of  cheapness  over  glass.  The 
microscopists  who  prefer  wood  tops  claim  that  glass  vessels  are 
easily  broken  on  the  slate  and  glass,  and  that  unless  extraordi- 
nary care  is  exercised  much  damage  will  result  from  this  source 
alone  in  the  laboratory.  This  objection  is  met,  however,  with 
the  statement  that  felt  pads  may  be  used  on  the  table  upon 
which  to  rest  the  delicate  glass,  and  the  neatness  in  appearance 
and  the  cleanliness  so  easily  produced  by  frequent  washing  are 
advantages  in  favor  of  slate  or  glass  not  to  be  despised  or 
overlooked. 

Photo-micrography.  —  An  adjoining  room  opening  into  the 
main  laboratory  should  be  fitted  up  for  experiments  in  photo- 
micrography. This  room  must  contain  the  following  furniture  : 

1.  A  long,  narrow  table  to  hold  the  camera  and  microscope 
and  attachments.     The  centre  of  the  room  is  the  best  position 
for  this  table. 

2.  A  table  to  contain  the  enlarging  camera  and  its  outfit. 

1  Jour.  App.  Mic.,  Vol.  II,  p.  231. 


312  APPENDIX 

3.  A  stand,  similar  to  a  camera  stand,  to  hold  the  lantern  and 
projection  apparatus.     This  stand  should  be  constructed  so  that 
the  top  can-  be  lowered,  elevated,  or  inclined  to  suit  the  char- 
acter of  the  picture  and  the  relative  position  of  the  lantern  and 
the  screen.     The  "  Continental  stand  "  will  be  found  well  adjusted 
for  this  purpose. 

4.  A  screen  sufficiently  large  to  hold  the  picture  projected 
from  the  lantern.     This  screen  may  be  constructed  on  rollers 
suspended  from  the  ceiling,  to  permit  of  being  rolled  up  as  a 
protection  against  dust,  and  to  remove  it  out  of  the  way  of  other 
operations. 

5.  The  windows  of  this  room  must  be  provided  with  opaque 
shades,  mounted  on  spring  rollers,  so  that  the  room  may  be 
darkened  in  a  few  moments  when  desired.     These  shades  should 
be  made  of  black  material  and  thick  enough  to  exclude  all  rays 
of  light.     The  best  way  to  attach  them  to  the  window  is  to  fasten 
the  roller  fixtures  on  the  sill  and  the  pulley  at  the  top  of  the 
window-frame.     The   cords,  after  leaving  the  pulleys,  are   run 
to  some  convenient  point  in  the  room,  where  they  will  be  out  of 
the  way  of  other  appliances  and  yet  so  situated  as  to  be  con- 
veniently reached.     The  rollers  should  be  placed  in  light-tight 
boxes,   and    around   the    window-frame   should   be   fastened    a 
grooved  frame,  in  which  the  edges  of  the  shade  will  run  when  it 
is  raised. 

6.  For  delicate  projection  direct  from  the  microscope  a  large 
ground-glass  plate  mounted  in  a  frame  will  be  found  necessary 
for  satisfactory  results. 

7.  .In  one  corner  of  this  room  a  dark  room  may  be  cut  off,  in 
which  to  perform  all  photographic  work  which  cannot  be  done 
in  white  light.     This  dark  room  should  be  provided  with  a  sink 
and  a  faucet  for  supplying  ample  water;    shelving  to  contain 
such  chemicals  as  are  affected  by  white  light ;   electric  lamps 
covered   by  ruby   globes ;    and  all  the  apparatus   required   to 
develop  the  sensitive  plates. 

8.  Storage  battery  to  supply  the  electricity  for  lighting  and 
heating. 


APPENDIX        .  313 

The  room  should  also  be  provided  with  the  following  addi- 
tional apparatus  and  conveniences  :  — 

1.  Earthenware  vessels  at  each  table  to  contain  the  refuse  and 
rejected  fluids.     These  vessels  should  be  emptied  every  day  by 
the  janitor  and  carefully  washed  clean  before  returning  to  the 
tables. 

2.  Carrying  trays  18"  X  20"  for  transporting  the.  bottles  and 
glassware  needed  for  the  students  during  the  hours  of  work. 
There  should  be  a  case  for  these  trays  near  the  shelves  contain- 
ing the  glassware  and  bottles  of  chemicals,  so  that  the  assistants 
may  conveniently  supply  the  wants  of  the  students  without  much 
trouble. 

3.  Dissecting  pans  with  wax,  cork,  or  paraffin  bottoms  will  be 
useful  for  all  work  in  zoology  and  where  injection  operations 
are  required. 

At  suitable  places  in  the  laboratory  should  be  located  tables 
for  holding  permanently  the  microtomes,  paraffin  and  celloidin 
apparatus,  water-baths,  and  card  indexes  of  the  material  belong- 
ing to  the  laboratory. 

In  the  laboratory  conducting  experiments  or  studies  in  subjects 
relating  to  zoological  matters,  an  aquarium  will  be  found  to  be 
a  valuable  apparatus,  to  contain  the  animals,  marine  and  fresh- 
water, in  a  live  condition  until  needed  for  the  experiment. 

Connected  with  the  laboratory  should  be  a  ventilating  hood 
for  carrying  off  all  noxious  and  poisonous  chemicals  used  for 
certain  operations. 

A  greenhouse  ought  to  be  an  adjunct  to  the  laboratory  in 
which  plant  studies  are  conducted,  but  if  this  is  not  possible, 
then  the  next  best  arrangement  will  be  window  trays  containing 
boxes  for  use  in  growing  plants.  These  trays  should  be  made 
of  zinc  to  catch  the  water  with  which  the  plants  are  watered, 
and  prevent  its  running  on  the  window-sill  and  on  to  the  floor 
of  the  laboratory. 

A  table  fo/r  holding  blast-lamps,  blowpipes,  and  such  like 
heating  apparatus  will  be  a  convenience,  and  sometimes  an 
absolute  necessity. 


314  APPENDIX 

A  library  attached  to  the  laboratory  has  become  one  of  the 
required  items  in  the  equipment.  If  the  institution  of  which 
the  laboratory  is  a  part  has  a  library,  even  then  it  is  the  part  of 
wisdom  to  transfer  to  an  adjoining  room  to  the  laboratory  those 
books  which  bear  directly  on  the  subjects  studied,  so  that  the 
workers  may  without  delay  or  inconvenience  refer  to  any  books 
for  information  desired. 


INDEX 


Abbe,  Prof.,  8,  16,  24. 

Abbe's  Substage  Illuminator,  15. 

Aberration,  Chromatic,  5. 

Absolute  Alcohol,  108,  288. 

Acetylene  Gas,  171. 

Acid,  Acetic,  73,  113,  241. 

Carminic,  107,  118. 

Chromic,  241. 

Chromic  Solution,  72. 

Glacial,  73,  107. 

Hydrochloric,  231. 

Lactic,  241. 

Nitric,  228. 

Osmic,  72,  237,  241. 

Salicylic,  287. 

Sulphuric,  84,  180,  190. 
^Equorea,  246. 
Agar-agar,  212. 
Air  bubbles  from  slides,  295. 
Albumen,  Mayer's,  70,  100. 
Alcohol,  76,  78,  261. 
Alum,  107,  113,  118,  190. 

Chrome,  180. 
American  Thread,  32. 
Ammonia  Carmine,  230. 
Ammonium  Chloride,  142. 
Amphioxus,  250. 
Amphipods,  248. 
Anaerobic  Culture,  220,  281. 

Kasparec's,  221, 

Novy's,  223. 

Wright's,  281. 
Angular  Aperture,  34. 
Anilin,  Green,  106. 
Anthozoa,  244. 
Aperture  Table,  34. 
Aplanatic,  5. 
Apochromatic,  8,  28. 
Apparatus  and  Accessories,  45. 
Asphalt,  129. 
Aubert,  A.  B.,  107^ 
Autoclave,  206. 


Bacteriology,  Apparatus  used  in,  202. 

Study  of,  200. 

Bardeen's  Freezing  Microtome.  93. 
Bausch  &  Lomb  Microtome,  46. 
Bausch  &  Lomb  Optical  Co.,  25,  29,  49, 

60,  162,  164. 

Beale's  Injection  Method,  231. 
Beck's  Automatic   Photographic    Lens, 

146. 

Belgian  Hone,  52. 
Bell  Glasses,  56. 
Benzol,  129. 

Bernhard's  Drawing  Desk,  136. 
Bismarck  Brown,  106,  120. 
Black  (Dead)  Color,  286. 
Black  Polish  for  Brass,  286. 
Bleaching,  225. 
Blood  Serum,  215. 
Blue  Stone  Hone,  146. 
Bopyrids,  248. 
Borax,  115. 
Bern's  Method,  237. 
Bouillon,  211. 

Box  for  Lantern  Slides,  195. 
Brachiopoda,  249. 
Brunswick  Black  Cement,  129. 
Brushes,  56. 
Bryozoa,  249. 
Bugula,  249, 

Cajal,  Ramon  y,  117. 
Camera,  the,  141. 

Cartridge  Kodak,  142. 

Long  Focus  Premo,  142. 

Large  Photo-micrographic,  Zeiss',  164. 
Camera  Lucida,  133. 
Camera   Method   for   making   Lantern 

Slides,  191. 
Camera  Tripod,  143. 
Campanularia,  246. 
Camphor  Water,  75. 
Canada  Balsam,  112. 


315 


INDEX 


Canada  Balsam,  Mounting  in,  127,  261. 

Carbutt's  Developing  Formulae,  267,  269. 

Carbutt's  Dry  Plates,  157,  178. 

Carbutt's  Fixing  Bath,  180. 

Carbutt's  Intensifying  Bath,  182. 

Care  of  Eyes,  43. 

Care  of  Lantern,  198. 

Carmine  Stain,  106. 

Celloidin  Imbedding,  66,  70. 

Celluloid  Films,  145. 

Cements,  127,  261,  295. 

Cerianthus,  245. 

Chaetopods,  248. 

Chloride  of  Gold,  188. 

Chromatic  Aberration,  5. 

Cirripedes,  248. 

Cladocera,  248. 

Clarifying,  80. 

Clark's  Lens,  146. 

Clavellina,  249. 

Cleaning  Fingers  of  Stains,  285. 

Cleaning  Formulae,  262. 

Cleaning  Glass,  260. 

Cleaning  Hands  of  Cement,  288. 

Cleaning  Negatives  of  Stains,  290. 

Clearing,  80. 

Clearing  Agents,  80. 

Anilin  Oil,  80. 

Bergamot  Oil,  80. 

Cedar  Oil,  80. 

Clove  Oil,  80. 

Origanum  Oil,  80. 

Turpentine  Oil,  80. 
Clove  Oil,  8,  80. 
Coarse  Adjustment,  12. 
Coddington  Lens,  2. 
Color  Screen,  How  to  make  a,  176. 
Colt's  Arc  Lamp,  168. 
Colt's  Lantern,  197. 
Comber,  T.,  173. 
Comparison    of    English    and    French 

Measures,  300. 
Compound  Staining,  no. 

Bismarck  Brown  —  Methyl  Green,  no. 

Carmine  —  Anilin  Blue,  no. 

Dahlia  —  Eosin,  no. 

Eosin  —  Methyl  Green,  no. 

Gentian  Violet —  Eosin,  no. 

Haematoxylin  —  Eosin,  no. 

Methyl  Violet — Eosin,  no. 

Safranin  —  Gentian,  no. 

Safranin  —  Haematoxylin,  no. 


Compensating  Ocular,  22. 

Compound  Microscope,  i,  9. 

Condensers,  16. 

Congo  Red,  112,  120. 

Contact    Method    for    Making   Lantern 

Slides,  191. 

Continental  Ocular,  21. 
Copepods,  248. 
Copper  Acetate,  75,  108. 
Copper  Chloride,  75,  108. 
Copper  Sulphate,  77,  242. 
Corals,  245. 

Corrosive  Sublimate,  261. 
Counting  Apparatus,  223. 
Cover-glass,  128. 
Cover-glass  Gauge,  29. 
Cover-glass,  Thickness,  28. 
Cramer's  Developer,  264,  268,  270. 
Cramer's  Dry  Plate,  158. 
Cramer's  Fixing  Bath,  271. 
Crown  Glass,  6. 
Crustacea,  248. 
Ctenophora,  247. 
Cucumaria,  247. 
Culture  Methods,  279. 
Cunina,  246. 

Cupric  Sulphate,  77,  242. 
Cutting  Glass,  296. 
Cutting  Paraffin  Mass,  66. 
Cutting  Sections,  84. 
Cyanin,  120,  142,  177. 

Dahlia,  no,  120. 

Dallmeyer's  Photographic  Lens,  146. 

Damar  Cement,  127,  261. 

Dark  Room,  183. 

Dark  Room  Equipment,  183. 

Darlot's  Photographic  Lens,  146. 

Decalcifying,  227,  293. 

Decapods,  248. 

Decapod  Cephalopods,  249. 

Degrees,  Tables  of,  304. 

Dehydrating,  78. 

Dehydrating  Apparatus,  Thomas's,  59. 

Delafield's  Haematoxylin,  69,  113. 

Developer  for  Lantern  Slides,  191. 

Developing  Formulae,  179,  263. 

Developing  Negative,  178. 

Developing  Pointers,  184. 

Developing  Trays,  150. 

Developing  Trays,  Sizes  Desirable,  150. 

Diamond  Ink,  288. 


INDEX 


317 


Diaphragm  Shutter,  148. 

Diaphragms,  Use  of,  44. 

Dilution  Method  of  Culture,  207, 

Diphyes,  247. 

Dissecting  Needles,  55. 

Dissecting  Scalpel,  55. 

Dissecting  Scissors,  55. 

Dissecting  Stand,  Zeiss',  3. 

Dissolving  Key,  198. 

Dissolving  Key  for  Oxy-hydrogen 
Lamp,  Colt's,  198. 

Dorids,  249. 

Double  Plate-holders,  148. 

Double  Staining.  See  Compound  Stain- 
ing. 

Doublets,  Wollaston's,  2. 

Drawing,  132. 

Drawing-desk,  Bernhard's,  136. 

Dry  Mounts,  131. 

Dry  Objective,  31. 

Dry  Plates,  173. 

Dry  Plates,  Orthochromatic,  175. 

Drying-racks  for  Negatives,  153. 

Eastman's  Developer,  264,  268. 
Eastman's  Plates,  158. 
Eastman's  Roll-holder,  144. 
Echinodermata,  247. 
Edwardsia,  245. 
Ehrlich's  Fluid,  77,  107. 
Ehrlich's  Haematoxylin,  113,  115. 
Ehrlich,  P.,  121. 
Eikonogen,  179. 
Eikonogen  Developer,  269. 
Elasmobranchs,  250. 
Electricity,  16. 
Electric  Lamps,  168. 
Elsching,  A.,  92. 
Embryos  of  Torpedo,  250. 
English  Thread,  32. 
Enlarging  Easel,  154. 
Entoniscids,  248. 
Eosin,  107. 

Equipment  for  Dark  Room,  183. 
Esmarch's  Roll  Culture,  220. 
Etching  Fluid,  287. 
Eucope,  246. 
Exposing  Limits,  177. 
Exposure  Meters,  154. 
Eyepiece  Micrometer,  27. 

Fine  Adjustment,  14.     . 
Fishes,  250. 


Fixing  Agents,  71. 

Alcohol,  72. 

Chromic  Acid  Solution,  72. 

Fleming's  Mixture,  73. 

Glacial  Acetic  Acid,  72. 

Hermann's  Solution,  73. 

Kleinenberg's  Formula,  74. 

Mayer's  Formula,  75. 

Mercuric  Chloride,  76. 

Merkel's  Solution,  74,  77. 

Nitric  Acid,  72. 

Perenyi's  Solution,  72. 

Platina  Chloride,  73. 

Schaffner's  Solution,  72. 
Fixing  and  Clearing    Baths,    180,    262, 

271. 

Fixing  Bath  Vessel,  151. 
Fixing  or  Killing,  71. 
Fixing  Sections  to  Glass  Slip,  100,  291. 
Fixing  Sections  to  Slip,  Huber's  Method, 
101. 

Eisen's  Method,  102. 

Koninski's  Method,  102. 

Weigert's  Method,  104. 
Flasks,  Erlenmeyer's,  59. 

Koch's,  59. 

Flemming's  Mixture,  73. 
Flint  Glass,  6. 
Focussing  Cloth,  148. 
Forceps,  54. 
Formaldehyde,  237. 
Formulae,  260,  263. 
Fractional  Culture,  218. 
Francais's    Extra    Rapid    Photographic 

Lens,  146. 

Freezing  Methods,  93. 
Fuerst's  Developer,  270. 
Furniture  of  Rooms,  307. 

Gas  Lamp,  171. 
Gas  Pressure  Regulators,  210. 
Gelatin,  215. 
Gelatin  Nutrient,  211. 
General  Stains,  105. 
Gentian  Neutral  Red,  120. 
Gentian  Violet,  107. 
Gephyreans,  248. 
Gerlach's  Formula,  106. 
German  Thread,  32. 
Glass,  Clearing,  260. 
Glass  Benches,  56. 
Glass  Slips,  128. 


318 


INDEX 


Glycerin,  113,  182. 

Glycerin  Jelly  Mounting,  131,  261,  262. 

Goerz  Lens,  146. 

Gold  Chlorid,  188. 

Golgi's  Method  for  Staining,  115, 116. 

Graduate,  151. 

Gram's   Method  for  Staining  Bacteria, 

118,  285. 

Grenacheri's  Borax-carmine,  69,  106. 
Ground-glass  Varnish,  290. 
Gundlach's  Photographic  Lens,  146. 

Haematein,  112. 

Haematoxylin,  112,  113. 

Halsted,  B.  D.,  288. 

Hammer  Dry  Plate,  158. 

Hammer's  Developer,  265. 

Hanging  Drop  Culture,  218. 

Hardening,  76. 

Haug's  Decalcifying  Method,  228. 

Heidenhain's  Iron  Hasmatoxylin  Stain, 

114. 

Hermann's  Formula,  73. 
Heteropods,  249. 
Hoffman's  Violet,  107. 
Holothurians,  247. 

Homogeneous  Immersion  Objective,  31. 
Huber,  G.  C.F  117. 
Huyghenian  Ocular,  21. 
Hydromedusae,  245. 
Hydroquinon,  179,  190. 
Hydroquin.on  Developer,  267. 

Iceland  Spar,  252. 

Illuminating  Apparatus,  15. 

Imbedding,  83. 

Imbedding  in  Celloidin,  89. 

Imbedding  in  Paraffin,  83. 

Immersion  Objective,  31. 

Incubator,  209. 

Index  of  Refraction,  296. 

Infiltration,  82. 

Injection,  229,  233. 

Ink  for  Writing  on  Glass,  286. 

Inoculation,  215. 

Intensifying  Solutions,  182,  272. 

"  Intra  vitam  "  Staining,  119. 

Iodine,  119. 

Iris  Diaphragm,  18. 

Iron  Black  Polish,  286. 

Iron  Sesquioxide,  231. 

Isopods,  248. 


James,  F.  L.,  26*. 
Jelinek,  O.,  Herr,  go. 
Jung,  R.,  97. 

Kaiserling's  Preserving  Solution,  29!. 
Killing,  Fixing,  and  Hardening,  71. 
Killing  for  Injection,  236. 
King's  Cement,  129. 
Kleinenberg's  Formula,  74,  247. 
Knauer,  F.,  Dr.,  261. 
Kodak,  142. 
Kodak,  Folding,  142. 
Kollicker,  von,  117. 
Kopsch,  Dr.,  118. 

Laboratory,  Arrangement  of,  305. 

Laboratory  Table,  307. 

Lacquering  Solution,  293. 

Lamellibranchs,  249. 

Lamp,  Carbutt's  Multum  in  Parvo,  149. 

Lamps  —  Electric,  Acetylene,  Gas,  Oxy- 

Hydrogen,  Oil,  165,  171. 
Lantern  and  its  Requisites,  195. 
Lantern,  Care  of,  198. 
Lantern,  Colt's   Criterion,  with  Electric 

Lamp    and    Microscope  Attachment, 

197. 

Lantern-slide  Developer,  191. 
Lantern  Slides,  How  to  make  them,  191. 
Leitz's  Microscope,  n. 
Lens,  Photographic,  146. 
Light,  Polarization  of,  252. 
Light-tight  Boxes  for  Sensitized  Plates, 

152. 

Lillie,  F.  R.,  62. 
Liriope,  246. 
List  of  Illustrations,  vii. 
Lithium  Carbonate,  115. 
Loxosoma,  249. 

Macerating  Fluid,  Schulze's,  288. 

Maceration,  237. 

Magnification,  138. 

Magnifiers,  Coddington,  2. 

Mann's  Method  for  Paraffin  Imbedding, 

86. 

Marine  Organisms,  Preservation  of,  240. 
Marlmann's  Preserving  Solution,  292. 
Marsh's  Bleaching  Method,  226. 
Mat  for  Lantern-slides,  194. 
Mayer,  Paul,  121. 
Mayer's  Formula,  75. 


INDEX 


319 


Mayer's  Method  for  Fixing  Sections  to 
Slip,  loo,  262. 

Camalum,  118. 

Haemalum,  112. 

Chlorine  Bleaching  Method,  226. 
Measures,  Tables  of,  299. 
Mechanical  Stage,  14. 
Merkel's  Solution,  74-77. 
Mercuric  Chloride,  76. 
Mercury  Bichloride,  76,  182. 
Metal,  190. 
Methyl  Blue,  120. 
Methyl  Green,  108. 
Methyl  Violet,  120. 
Metric  Measures,  Table  of,  299. 
Micrometer,  27. 
Micrometer  Eyepiece,  27. 
Micrometer,  Filar,  27. 
Micrometer  Screw,  32. 
Micrometer,  Ocular,  27. 
Micron,  28. 

Microscope  Attachment  for  Lantern,  197. 
Microscope,  Compound,  i,  9. 
Microscope,  Continental,  7. 
Microscope,  How  to  use,  41. 
Microscope,  Single  or  Dissecting,  i,  3. 
Microscope,  Spencer,  13. 

Leitz,  ii. 
Microscope     Stand     with    Mechanical 

Stage,  Frontispiece. 
Microtome,  Bausch  &  Lomb,  46. 

Bardeen's  Freezing,  93. 

Freezing,  93. 

Hand,  45. 

How  to  care  for,  52. 

Jung's,  97. 

Reichert's,  50. 

Ribbon  Carrier,  49. 
Microtome  Knife,  52. 
Microtome,  Minot's  Precision,  49. 
Microtome  Sharpening  Knife,  52. 
Miller's  Scheme  for  Completing  Mount, 

69. 

Miller,  W.  S.,  61. 

Minot's  Automatic  Microtome,  48. 
Molgula,  249. 
Moll,  J.  W.,  86. 
Mollusca,  249. 
Moore's  Staining-dish,  109. 
Monochromatic  Filter,  275. 
Mono-refringent  Light,  256. 
Morrison's  Photographic  Lens,  146. 


Mortars,  59. 

Mounting  in  Canada  Balsam,  123. 
Mounting  Media,  261. 
Miiller's  Solution,  115. 

Naples  Staining-jar,  109. 
Needle-holders,  55. 
Negative  Drying  Rack,  153. 
Negative  Titling,  297. 
Negative  Washing  Box,  151. 
Negatives,  Defects  in,  184. 
Nemathelminths,  247. 
Nemerteans,  248. 
New  York  Plates,  158. 
Nitrate  of  Silver,  116. 
Nivellating  Apparatus,  220. 
Nose-piece,  Revolving,  19, 
Novey,  F.  G.,  211. 
Novey's  Thermo-regulator,  210. 
Nuclear  Stains,  in. 
Numerical  Aperture,  33. 
Nutrient  Media,  211. 

Obelia,  246. 
Objectives,  30. 

Achromatic,  36. 

Apochromatic,  36. 
List  of,  40. 
Magnification  of,  40. 

Dry,  31. 

Homogeneous,  31. 

Immersion,  31. 
Oculars,  21. 

Compensating,  22. 

Continental,  21. 

Huyghenian,  21. 

Micrometer,  27. 

Projection,  25,  26. 

Ramsden's  Positive,  22. 
Oil,  Anilin,  80,  107,  118. 

Bergamot,  81. 

Cedar,  81. 

Cloves,  8 1. 

Origanum,  81. 

Turpentine,  81. 
Opisthobranchs,  249. 
Orienting,  98. 

Eyclesheimer's  Method,  100. 

Woodworth's  Method,  98. 
Orthochromatic  Plates,  175. 
Orthochromatizing  Plates,  175. 
Ostropods,  248. 


320 


INDEX 


Palm  Oil  Soap,  53. 

Paper  Imbedding  Box,  64. 

Paraffin  Imbedding,  66,  70. 

Paraffin  Imbedding  Box,  64. 

Paste,  294. 

Pedicellina,  249. 

Perenyi's  Formula,  72. 

Perophora,  249. 

Petit's  Solution,  75,  108. 

Petri  Dishes,  57,  219. 

Phloroglucin,  228. 

Photographic  Lenses,  146. 

Photo-micrographs,  156. 

Photo-micrographic  Apparatus,  141,  159, 

3"- 

Photo-micrographic  Projection  Appara- 
tus, Zeiss',  164. 
Photo-micrographs,  How  to  make  them, 

141,  156. 
Physalia,  246. 
Picric  Acid,  74. 
Picric  Alcohol,  74.     • 
Pipettes,  58. 
Plate  Culture,  219. 
Platinic  Chloride,  73. 
Polariscope  Adjustment,  255. 
Polarization  of  Light,  252. 
Polarizer,  Zeiss',  254. 
Polarizers,  Mohl's  Investigations  of,  256. 
Pollen  Tube  Emission,  288. 
Positives,  Making  the,  186. 
Potassium    Bicarbonate,    77,    116,    179, 
242. 

Bromide,  179,  190. 

Ferricyanide,  115,  231. 

Iodine,  119. 

Potato  Culture  Method,  215,  282. 
Premo  Camera,  142. 
Preparation    of    Tissue    for    Mounting, 

66. 

Preserving  Media  for  Plants,  263. 
Printing  Formulae,  277. 
Printing  Frame,  152. 
Projection  Ocular,  26. 
Prosobranchs,  249. 
Przesmycki,  A.  M.,  121. 
Pteropods,  249. 
Pyenogcnids,  248. 
Pyrogallic  Acid  Developer,  264. 

Stain  removed  from  Hands,  285. 

Queen's  Pantagraph  Lens,  146. 


Ramsden's  Positive  Ocular,  22. 
Ranvier's  Picro-carmine  Stain,  107. 
Ravenel,  M.  P.,  212. 
Ray  Filter,  150. 

Reduction  of  Intense  Negatives,  274. 
Reeves'  Bath,  62. 
Refraction,  Index  of,  296. 
Refraction  of  Light,  252. 
Reichert's  Thermo-regulator,  300. 
Retouching  Frame,  153. 

the  Negative,  297. 
Revolving  Nose-piece,  19. 
Rheostat,  Colt's  Adjustable,  169. 
Ribbon  Sections,  49,  84. 
Ripart's  Solution,  75,  107. 
Roll-holder,  144. 
Room  and  its  Furniture,  305. 
Rosen's  Method  for  Imbedding,  89. 
Ross'  Photographic  Lens,  146. 

Safranin  Stain,  108. 

Sagitta,  248. 

Salicylate  of  Soda,  100. 

Salpae,  250. 

Scalpels,  55. 

Schizopods,  248. 

Schott,  Dr.,  8. 

Scissors,  55. 

Screen,  Color,  176. 

Scyphomedusae,  246. 

Secondary  or  Non-nuclear  Stains,  in. 

Section-lifter,  56. 

Seed  Plate  Developer  and  Fixing  Baths, 

266,  267,  269. 
Seed's  Dry  Plate,  158. 
Selective  Stains,  105. 
Selenite,  256. 
Serpulidae,  244. 
Shellac,  129. 
Silvering  Glass,  289. 
Silver  Nitrate,  116. 

Silver  Stains  removed  from  Hands,  285. 
Siphonophores,  246. 
Size  of  Photographic  Plates,  290. 
Sliding  Objective  Changers,  20. 
Society  Screw,  32. 
Soda,  Carbonate,  179,  190. 

Sulphite  Crystals,  179,  180,  190. 

Hyposulphite,  180,  188. 
Sodium  Acetate,  188. 
Special  Injection  Masses,  232. 
Spherical  Aberration,  4. 


INDEX 


321 


Spring  Clips,  56. 
Stage,  Mechanical,  14. 
Stage,  Microscope,  14. 
Stain,  How  to,  109. 
Stains,  105,  282. 

(See  also  under  each  separate  stain.) 
Stanley  Dry  Plate,  158. 
Stanley's  Developer,  266. 
Starch  Injection,  231. 
Steinheil's  Photographic  Lens,  146. 
Sterilizer,  Hot  Air,  203. 

Steam,  205. 
Sterilizing,  205. 
Sterling's  Constant  Pressure   Injection, 

235- 

Stoppers,  Loosening,  296. 
Strasser's  Method,  239. 
Suter's  Photographic  Lens,  146. 
Swing-out  Condenser,  Zeiss',  17. 
Synopta,  247. 

Syracuse  Watch  Glasses,  58. 
Syringe,  Injection,  234. 

Table  of  Contents,  2. 
Tables,  Useful,  260. 
Table-top  Finish,  298,  310. 
Thermo-regulators,  210,  230. 
Thickness  of  Cover-glass,  28. 
Thyone,  247. 

Tray,  Hard  Rubber  Developing,  150. 
Trematodes,  247. 


Tube  Culture,  216. 
Tube  Length,  10. 
Tunicates,  249. 
Turbellaria,  247. 
Turn-table,  60. 
Turpentine,  80. 

Victoria  Blue  Stain,  108. 
Virodtzeff  s  Preserving  Solution,  291. 
Velella,  246. 
Vermes,  247. 

Wash  Bottles,  58. 

Watch-glasses,  58. 

Water-baths,  61. 

Weigert's    Stain    for    Nervous    System, 

114. 

Welsbach's  Gas  Burner,  164. 
Wickersheim's  Preserving  Solution,  291. 
Wire  Baskets,  209. 
Wollaston's  Doublets,  2. 
Wright's  Culture  Method,  281. 

Xylol,  70,  129. 

Zeiss,  8. 

Zeiss'  Anastigmat  Photographic  Lens, 
146. 

Zeiss'  Compound  Microscope,  Frontis- 
piece. 

Zeiss'  Dissecting  Microscope,  3. 


A  TEXT-BOOK  OF   ZOOLOGY 

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OVERDUE. 


AU6    11  I! 


FEB    21 


FEB  23  1839 

AUG  1  1  1950 
OCT  2  *  1959 

Oc10'59KAL 
MAY  2  6  I960 


JUL  1  2  I960 


JUL  1  2  1960 

NQV  1  5  1962 
VNo'62DP 


LD  21-f 


LjJ 


U.C.  BERKELEY  LIBRARIES 


COEbObMSSO 


